Audited DRAFT Final Report

METABOLISM AND PHARMACOKINETICS OF DIISOPROPYL ETHER IN MALE AND FEMALE RATS: PILOT STUDY

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-934

RTI Study Code:	Rt05-934

RTI Report No.	RTI/0209408.001

March 24December 10, 2013January 11, 2015

Timothy R. Fennell, Ph.D.

Study Director

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

SUMMARY

This study evaluated the pharmacokinetics and disposition of diisopropyl ether (DIPE) in male and Fischer 344 rats following nose only exposure.  Three separate studies were conducted.

In Study A, nose-only inhalation exposure of jugular-vein cannulated male and female Fischer 344 rats to 3600 ppm DIPE for 6 hours (actual concentration 3643 ± 177 ppm) was conducted, and serial blood samples were obtained up to 16 24 h following exposure.  Headspace GC-MS analysis of DIPE, isopropanol and acetone was conducted on the blood samples.  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 16 hr. In the male rats, isopropanol was below LOQ in preexposure samples, all three samples collected at 5 min, and in one of the three collected at 10 min.  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 16 hr.  In the female rats, isopropanol was below LOQ in preexposure samples, and all samples collected at 5, 10, and 15 min.  The concentration of isopropanol rose steadily during the exposure, and then declined gradually.  In male rats, blood concentrations of isopropanol peaked at approximately twice those of female rats.  All samples were below LOQ by 16 hr.  In male and female rats, acetone was above LOQ in 5 of the 6 preexposure samples.  Acetone levels in both male and female rats rose steadily during the exposure, and in males continued to rise following exposure until 8 hr following the initiation of exposure.  In females, the acetone levels were approximately the same between 6 and 8 hours following the exposure, and then declined rapidly reaching preexposure levels by 16 hrs in females.  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,  and for isopropanol the peak concentrations in males were almost twice those in females .  Male rats reached preexposure levels by 24 hours.  Non-compartmental pharmacokinetic analysis of  the mean concentrations of each analyte was conducted using WinNonlin.  DIPE was eliminated with a half life of Pharmacokinetic analysis of DIPE indicated a half life of 0.92 h in male rats, and 0.76 h in female rats.  Isopropanol had an apparent half life of 1.16 and 1.23 h in male and female rats, respectively.  Acetone had an apparent half life of 2.31 and 2.32 h in male and female rats, respectively.

In Study B, four male Fischer 344 rats were exposed to [2-14C] diisopropyl ether in a nose-only inhalation system at a target concentration of 3600 ppm for 6 hours (actual concentration 3529 ( 40 ppm).  Following exposure, the 4 rats were euthanized immediately and the radioactivity in the carcass was determined by digestion and scintillation counting (Group 2, Study B).  In Study C, four male Fischer 344 rats were exposed to a mixture of 13C6 and [2-14C] diisopropyl ether in a nose-only inhalation system at a target concentration of 3600 ppm for 6 hours (actual concentration 3334 ( 99 ppm, actual exposure time 5 hours and 40 minutes).  The rats were transferred to all-glass metabolism cages, and urine, feces, exhaled CO2 and exhaled volatiles were collected for seven days (Group 3, Study C).  After seven days, the rats were euthanized and the radioactivity remaining in the carcass was determined by digestion and scintillation counting.  Radioactivity was eliminated primarily as exhaled volatiles (70 % of the recovered radioactivity).  Approximately 20 % was recovered as 14CO2. Small amounts of radioactivity were recovered in urine (5% of the recovered radioactivity).  A lesser amount was recovered (1%) in feces, and 2.6% in carcass at 7 days.

Based on the recovered radioactivity, the estimated dose in the rats in Group 2 was 371 373 ( 8 mg/kg, and in Group 2 3 was 299 318 ( 7 8 mg/kg.  Elimination of radioactivity was rapid, with 93.7% and 95.5% of the radioactivity recovered within 24 h and 48 h, respectively, following the end of exposure.

The objectives of the study to develop a system for inhalation exposure to DIPE and rr to develop analytical methods for the analysis of DIPE, acetone and isopropanol were achieved.  Additional objectives achieved were characterization of the kinetics of DIPE, acetone and isopropanol in blood from rats exposed to 3600 ppm DIPE by inhalation for 6 hours, and  analysis of the disposition of radioactivity conducted following exposure to [2-14C] DIPE, and 13C6 and [2-14C] DIPE.  The objective of characterizing urinary metabolites was not pursued because of the low excretion of radioactivity in urine.

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 prestudy exposure system method development was completed at CIIT Centers for Health Research and was not conducted under GLPs.  The analysis of port to port variability of the nose only inhalation system was conducted as part of this prestudy conduct, and was not repeated as part of the GLP verification of system performance.

The method of synthesis of the test chemical diisopropyl ether and [2-14C]-diisopropyl ether, or their location was not available at the time of conduct of the study.   Information on the method of synthesis of [2-14C]-diisopropyl ether has been obtained from the vendor.  However, the The suppliers of unlabeled diisopropyl ether has have been unable to provide information on the methods 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.  Specimens, as applicable, raw data, copies of the inhalation data, and the final report generated as a result of this study are archived at RTI International.

Timothy R. Fennell, Ph.D.		Date

Study Director

Derek Swick  Ph.D.		Date

Sponsors Representative

Table of Contents

Page

  TOC \o "3-3" \h \z \t "Heading 1,1,Heading 2,2"    HYPERLINK \l "_Toc351999782"  1.0  	Introduction	  PAGEREF _Toc351999782 \h  1

  HYPERLINK \l "_Toc351999783"  2.0  	Objectives and Protocol	  PAGEREF _Toc351999783 \h  1

 HYPERLINK \l "_Toc351999784" 3.0  	Materials and Methods	  PAGEREF _Toc351999784 \h  221

 HYPERLINK \l "_Toc351999785" 3.1  	Test Substance	  PAGEREF _Toc351999785 \h  221

  HYPERLINK \l "_Toc351999786"  3.1.1  	Nonradiolabeled diisopropyl ether	  PAGEREF _Toc351999786 \h  2

  HYPERLINK \l "_Toc351999787"  3.1.2  	(U-13C6)-labeled diisopropyl ether	  PAGEREF _Toc351999787 \h  2

  HYPERLINK \l "_Toc351999788"  3.1.2  	[2-14C]-labeled diisopropyl ether	  PAGEREF _Toc351999788 \h  2

  HYPERLINK \l "_Toc351999789"  3.2  	Reference Substances	  PAGEREF _Toc351999789 \h  3

  HYPERLINK \l "_Toc351999790"  3.3  	Test System	  PAGEREF _Toc351999790 \h  3

  HYPERLINK \l "_Toc351999791"  3.4  	Test Chemical Preparation and Analysis	  PAGEREF _Toc351999791 \h  4

  HYPERLINK \l "_Toc351999792"  3.5  	Inhalation Exposure	  PAGEREF _Toc351999792 \h  5

  HYPERLINK \l "_Toc351999793"  3.5.1 Exposure Analysis	  PAGEREF _Toc351999793 \h  6

  HYPERLINK \l "_Toc351999794"  3.6  	Collection of Samples	  PAGEREF _Toc351999794 \h  6

  HYPERLINK \l "_Toc351999795"  3.6.1	Blood Sample Collection	  PAGEREF _Toc351999795 \h  6

 HYPERLINK \l "_Toc351999796" 3.7.1	Excreta	  PAGEREF _Toc351999796 \h  776

  HYPERLINK \l "_Toc351999797"  3.6.2	Carcass	  PAGEREF _Toc351999797 \h  7

  HYPERLINK \l "_Toc351999798"  3.6.2	Tissues	  PAGEREF _Toc351999798 \h  7

  HYPERLINK \l "_Toc351999799"  3.8  	Analysis of Blood Samples for DIPE, Isopropanol, and Acetone	  PAGEREF _Toc351999799 \h  8

  HYPERLINK \l "_Toc351999800"  3.8  	Analysis of Samples for Total Radioactivity	  PAGEREF _Toc351999800 \h  8

  HYPERLINK \l "_Toc351999801"  3.8.1	Excreta	  PAGEREF _Toc351999801 \h  98

  HYPERLINK \l "_Toc351999802"  3.8.2	Tissues and Carcass	  PAGEREF _Toc351999802 \h  9

  HYPERLINK \l "_Toc351999803"  3.8.3  Exhaled Breath Traps	  PAGEREF _Toc351999803 \h  9

  HYPERLINK \l "_Toc351999804"  3.8.4  Exhaled Volatiles Traps	  PAGEREF _Toc351999804 \h  9

  HYPERLINK \l "_Toc351999805"  3.9  	Data Collection and Reporting	  PAGEREF _Toc351999805 \h  9

  HYPERLINK \l "_Toc351999806"  4.0  	Results	  PAGEREF _Toc351999806 \h  10

  HYPERLINK \l "_Toc351999807"  4.1  	Analysis of Radiolabeled Test Chemical Identity and Purity	  PAGEREF _Toc351999807 \h  10

  HYPERLINK \l "_Toc351999808"  4.2  	Test Chemical Formulation	  PAGEREF _Toc351999808 \h  10

  HYPERLINK \l "_Toc351999809"  4.3  	Inhalation exposure	  PAGEREF _Toc351999809 \h  10

 HYPERLINK \l "_Toc351999810" 4.4  	DIPE, Acetone and Isopropanol in Blood During and  Following Exposure	  PAGEREF _Toc351999810 \h  111110

  HYPERLINK \l "_Toc351999811"  4.5  	Radioactivity Retained Following Exposure	  PAGEREF _Toc351999811 \h  12

  HYPERLINK \l "_Toc351999812"  4.6  	Excretion of Radioactivity	  PAGEREF _Toc351999812 \h  12

  HYPERLINK \l "_Toc351999813"  5.0  	Discussion	  PAGEREF _Toc351999813 \h  13

 HYPERLINK \l "_Toc351999814" 6.0  	References	  PAGEREF _Toc351999814 \h  151514

 HYPERLINK \l "_Toc351999815" 7.0  	RECORDS and Reports	  PAGEREF _Toc351999815 \h  151514

 HYPERLINK \l "_Toc351999816" 8.0  	Storage of Records and Biological Samples	  PAGEREF _Toc351999816 \h  151514

 HYPERLINK \l "_Toc351999817" 9.0  	REGULATORY COMPLIANCE	  PAGEREF _Toc351999817 \h  161615

 HYPERLINK \l "_Toc351999818" 10.0  	STUDY PARTICIPANTS	  PAGEREF _Toc351999818 \h  161615

 List of Tables

Page

  TOC \h \z \t "Table Title" \c "Table"    HYPERLINK \l "_Toc419387375"  Table 1.	  Male and Female Fischer 344 Rats Used on the Study	  PAGEREF _Toc419387375 \h  17

  HYPERLINK \l "_Toc419387376"  Table 2.	  Exposure Conditions and Concentration of DIPE, Study A.	  PAGEREF _Toc419387376 \h  18

  HYPERLINK \l "_Toc419387377"  Table 3.	  Exposure Conditions and Concentration of [2-14C]DIPE/DIPE, Study B.	  PAGEREF _Toc419387377 \h  19

  HYPERLINK \l "_Toc419387378"  Table 4.	  Exposure Conditions and Concentration of [2-14C]DIPE/13CDIPE, Study C.	  PAGEREF _Toc419387378 \h  20

  HYPERLINK \l "_Toc419387379"  Table 5.	  Concentration of DIPE in blood from male and female rats exposed to 3600 ppm DIPE.	  PAGEREF _Toc419387379 \h  21

  HYPERLINK \l "_Toc419387380"  Table 6.	  Concentration of Isopropanol in blood from male and female rats exposed to 3600 ppm DIPE.	  PAGEREF _Toc419387380 \h  22

  HYPERLINK \l "_Toc419387381"  Table 7.	  Concentration of Acetone in blood from male and female rats exposed to 3600 ppm DIPE.	  PAGEREF _Toc419387381 \h  23

  HYPERLINK \l "_Toc419387382"  Table 8.	  Pharmacokinetic Analysis of DIPE, Isopropanol and Acetone in male and female rats exposed to 3600 ppm DIPE.	  PAGEREF _Toc419387382 \h  24

  HYPERLINK \l "_Toc419387383"  Table 9.	  Total Recoverya of Radioactivity following Exposure to 3600 ppm [2-14C]DIPE/DIPE	  PAGEREF _Toc419387383 \h  25

  HYPERLINK \l "_Toc419387384"  Table 10.	  Recovery of Radioactivity in Carcass following Exposure to 3600 ppm [2-14C]DIPE/DIPE (Group 2)	  PAGEREF _Toc419387384 \h  26

  HYPERLINK \l "_Toc419387385"  Table 11.	  Recovery of Radioactivity in Excreta and Carcass following Exposure to 3600 ppm [2-14C]DIPE/13C6DIPE (Group 3)	  PAGEREF _Toc419387385 \h  27

  HYPERLINK \l "_Toc419387386"  Table 12.	  Recovery of Radioactivity in Urine following Exposure to 3600 ppm [2-14C]DIPE/13C6DIPE (Group 3)	  PAGEREF _Toc419387386 \h  28

  HYPERLINK \l "_Toc419387387"  Table 13.	  Recovery of Radioactivity in Feces following Exposure to 3600 ppm [2-14C]DIPE/13C6DIPE (Group 3)	  PAGEREF _Toc419387387 \h  29

  HYPERLINK \l "_Toc419387388"  Table 14.	  Recovery of Radioactivity in CO2 Trap 1  following exposure to 3600 ppm [2-14C]DIPE/13C6DIPE (Group 3)	  PAGEREF _Toc419387388 \h  30

  HYPERLINK \l "_Toc419387389"  Table 15.	  Recovery of Radioactivity in CO2 Trap 2 following exposure to 3600 ppm [2-14C]DIPE/13C6DIPE (Group 3)   PAGEREF _Toc419387389 \h  31

  HYPERLINK \l "_Toc419387390"  Table 16.	  Recovery of Radioactivity in Exhaled VOC Trap 1 following exposure to 3600 ppm [2-14C]DIPE/13C6DIPE (Group 3)	  PAGEREF _Toc419387390 \h  32

  HYPERLINK \l "_Toc419387391"  Table 17.	  Recovery of Radioactivity in Exhaled VOC Trap 2 following Exposure to 3600 ppm [2-14C]DIPE/13C6DIPE (Group 3)	  PAGEREF _Toc419387391 \h  33

  HYPERLINK \l "_Toc419387392"  Table 18.	  Cumulative Recovery of Radioactivity in Excreta and Carcass following Exposure to 3600 ppm [2-14C]DIPE/13C6DIPE (Group 3)	  PAGEREF _Toc419387392 \h  34

  HYPERLINK \l "_Toc419387393"  Table 19.	  Recovery of Radioactivity in Tissues from Rat CM-02 following Exposure to 3600 ppm [2-14C]DIPE/13C6DIPE (Group 3)	  PAGEREF _Toc419387393 \h  35

  HYPERLINK \l "_Toc419387394"  Table 20.	  Normalized Recovery of Radioactivity in Exposure B and C (Groups 2 and 3).	  PAGEREF _Toc419387394 \h  36

  HYPERLINK \l "_Toc419387395"  Sample Naming and Abbreviations	  PAGEREF _Toc419387395 \h  2

  HYPERLINK \l "_Toc419387396"  In the tables of raw data values in this appendix, the table headings are derived from the conventions of sample naming used in the Debra( system.  These are explained below.  Example calculations and formulae are provided in Appendix G.	  PAGEREF _Toc419387396 \h  2

  HYPERLINK \l "_Toc419387397"  Table 1.  Preparation of 14C DIPE Formulation (Feedstock) for Inhalation Exposure	  PAGEREF _Toc419387397 \h  4

  HYPERLINK \l "_Toc419387398"  14C DIPE for Study B	  PAGEREF _Toc419387398 \h  4

  HYPERLINK \l "_Toc419387399"  Table 1 (contd).  Preparation of 14C DIPE Formulation (Feedstock) for Inhalation Exposure	  PAGEREF _Toc419387399 \h  5

  HYPERLINK \l "_Toc419387400"  13C/14C DIPE For Study C	  PAGEREF _Toc419387400 \h  5

  HYPERLINK \l "_Toc419387401"  Table 2.  Recovery of Radioactivity in Samples from Group B.	  PAGEREF _Toc419387401 \h  6

  HYPERLINK \l "_Toc419387402"  Table 3.  Total Recovery of Radioactivity in Samples from Group B.	  PAGEREF _Toc419387402 \h  7

  HYPERLINK \l "_Toc419387403"  Table 4.  Recovery of Radioactivity in Urine from Group C.	  PAGEREF _Toc419387403 \h  8

  HYPERLINK \l "_Toc419387404"  Table 4 (contd).  Recovery of Radioactivity in Urine from Group 2.	  PAGEREF _Toc419387404 \h  9

  HYPERLINK \l "_Toc419387405"  Table 5.  Recovery of Radioactivity in Feces from Group C.	  PAGEREF _Toc419387405 \h  10

  HYPERLINK \l "_Toc419387406"  Table 5 (contd).  Recovery of Radioactivity in Feces from Group C.	  PAGEREF _Toc419387406 \h  11

  HYPERLINK \l "_Toc419387407"  Table 6.  Recovery of Radioactivity in CO2 Trap 1 from Group C.	  PAGEREF _Toc419387407 \h  12

  HYPERLINK \l "_Toc419387408"  Table 6 (contd).  Recovery of Radioactivity in CO2 Trap 1 from Group C.	  PAGEREF _Toc419387408 \h  13

  HYPERLINK \l "_Toc419387409"  Table 7.  Recovery of Radioactivity in CO2 Trap 2 from Group C.	  PAGEREF _Toc419387409 \h  14

  HYPERLINK \l "_Toc419387410"  Table 7 (contd).  Recovery of Radioactivity in CO2 Trap 2 from Group C.	  PAGEREF _Toc419387410 \h  15

  HYPERLINK \l "_Toc419387411"  Table 8.  Recovery of Radioactivity in Exhaled VOC Trap 1 from Group C.	  PAGEREF _Toc419387411 \h  16

  HYPERLINK \l "_Toc419387412"  a Samples from CM-04 were not analyzed.	  PAGEREF _Toc419387412 \h  16

  HYPERLINK \l "_Toc419387413"  * in LOD column indicates that sample DPM were less than background DPM, and in LOQ column indicates that sample DPM was less than 3 x background. Table 8 (contd).  Recovery of Radioactivity in Exhaled VOC Trap 1 from Group C.	  PAGEREF _Toc419387413 \h  16

  HYPERLINK \l "_Toc419387414"  Table 8 (contd).  Recovery of Radioactivity in Exhaled VOC Trap 1 from Group C.	  PAGEREF _Toc419387414 \h  17

  HYPERLINK \l "_Toc419387415"  a Samples from CM-04 were not analyzed.	  PAGEREF _Toc419387415 \h  17

  HYPERLINK \l "_Toc419387416"  Table 9.  Recovery of Radioactivity in VOC Trap 2 from Group C.	  PAGEREF _Toc419387416 \h  18

  HYPERLINK \l "_Toc419387417"  Table 9 (contd).  Recovery of Radioactivity in VOC Trap 2 from Group C.	  PAGEREF _Toc419387417 \h  19

  HYPERLINK \l "_Toc419387418"  Table 10.  Recovery of Radioactivity in Nose Only Tube Rinse from Group C.	  PAGEREF _Toc419387418 \h  20

  HYPERLINK \l "_Toc419387419"  Table 11.  Recovery of Radioactivity in Cage Rinse from Group C.	  PAGEREF _Toc419387419 \h  20

  HYPERLINK \l "_Toc419387420"  Table 14.  Recovery of Radioactivity in Carcass Digest from Group C.	  PAGEREF _Toc419387420 \h  21

  HYPERLINK \l "_Toc419387421"  Table 15.  Total Recovery of Radioactivity in Samples from Group C.	  PAGEREF _Toc419387421 \h  22

  HYPERLINK \l "_Toc419387422"  Table 15 (contd).  Total Recovery of Radioactivity in Samples from Group C.	  PAGEREF _Toc419387422 \h  23

  HYPERLINK \l "_Toc419387423"  Table 15 (contd).  Total Recovery of Radioactivity in Samples from Group C.	  PAGEREF _Toc419387423 \h  24

  HYPERLINK \l "_Toc419387424"  Table 15 (contd).  Total Recovery of Radioactivity in Samples from Group C.	  PAGEREF _Toc419387424 \h  25

  HYPERLINK \l "_Toc419387425"  Table 15 (contd).  Total Recovery of Radioactivity in Samples from Group C.	  PAGEREF _Toc419387425 \h  26

Table 1.	  Male and Female Fischer 344 Rats Used on the Study

Table 2.	  Exposure Conditions and Concentration of DIPE, Study A.

Table 3.	  Exposure Conditions and Concentration of [2-14C]DIPE/DIPE, Study B.

Table 4.	  Exposure Conditions and Concentration of [2-14C]DIPE/13CDIPE, Study C.

Table 5.	  Concentration of DIPE in blood from male and female rats exposed to 3600 ppm DIPE.

Table 6.	  Concentration of Isopropanol in blood from male and female rats exposed to 3600 ppm DIPE.

Table 7.	  Concentration of Acetone in blood from male and female rats exposed to 3600 ppm DIPE.

Table 8.	  Pharmacokinetic Analysis of DIPE, Isopropanol and Acetone in male and female rats exposed to 3600 ppm DIPE.

Table 9.	  Total Recoverya of Radioactivity following Exposure to 3600 ppm [2-14C]DIPE/DIPE

Table 10.	  Recovery of Radioactivity in Carcass following Exposure to 3600 ppm [2-14C]DIPE/DIPE (Group 2)

Table 11.	  Recovery of Radioactivity in Excreta and Carcass following Exposure to 3600 ppm [2-14C]DIPE/13C6DIPE (Group 3)

Table 12.	  Recovery of Radioactivity in Urine following Exposure to 3600 ppm [2-14C]DIPE/13C6DIPE (Group 3)

Table 13.	  Recovery of Radioactivity in Feces following Exposure to 3600 ppm [2-14C]DIPE/13C6DIPE (Group 3)

Table 14.	  Recovery of Radioactivity in CO2 Trap 1  following exposure to 3600 ppm [2-14C]DIPE/13C6DIPE (Group 3)

Table 15.	  Recovery of Radioactivity in CO2 Trap 2 following exposure to 3600 ppm [2-14C]DIPE/13C6DIPE (Group 3)

Table 16.	  Recovery of Radioactivity in Exhaled VOC Trap 1 following exposure to 3600 ppm [2-14C]DIPE/13C6DIPE (Group 3)

Table 17.	  Recovery of Radioactivity in Exhaled VOC Trap 2 following Exposure to 3600 ppm [2-14C]DIPE/13C6DIPE (Group 3)

Table 18.	  Cumulative Recovery of Radioactivity in Excreta and Carcass following Exposure to 3600 ppm [2-14C]DIPE/13C6DIPE (Group 3)

Table 19.	  Recovery of Radioactivity in Tissues from Rat CM-02 following Exposure to 3600 ppm [2-14C]DIPE/13C6DIPE (Group 3)

Table 20.	  Normalized Recovery of Radioactivity in Exposure B and C (Groups 2 and 3).

Sample Naming and Abbreviations

In the tables of raw data values in this appendix, the table headings are derived from the conventions of sample naming used in the Debra( system.  These are explained below.  Example calculations and formulae are provided in Appendix G.

Table 1.  Preparation of 14C DIPE Formulation (Feedstock) for Inhalation Exposure

14C DIPE for Study B

Table 1 (contd).  Preparation of 14C DIPE Formulation (Feedstock) for Inhalation Exposure

13C/14C DIPE For Study C

Table 2.  Recovery of Radioactivity in Samples from Group B.

Table 3.  Total Recovery of Radioactivity in Samples from Group B.

Table 4.  Recovery of Radioactivity in Urine from Group C.

Table 4 (contd).  Recovery of Radioactivity in Urine from Group 2.	10

Table 5.  Recovery of Radioactivity in Feces from Group C.	11

Table 5 (contd).  Recovery of Radioactivity in Feces from Group C.	12

Table 6.  Recovery of Radioactivity in CO2 Trap 1 from Group C.	13

Table 6 (contd).  Recovery of Radioactivity in CO2 Trap 1 from Group C. 14

Table 7.  Recovery of Radioactivity in CO2 Trap 2 from Group C.	15

Table 7 (contd).  Recovery of Radioactivity in CO2 Trap 2 from Group C. 16

Table 8.  Recovery of Radioactivity in Exhaled VOC Trap 1 from Group C. 17

a Samples from CM-04 were not analyzed.	17

* in LOD column indicates that sample DPM were less than background DPM, and in LOQ column indicates that sample DPM was less than 3 x background. Table 8 (contd).  Recovery of Radioactivity in Exhaled VOC Trap 1 from Group C.	17

a Samples from CM-04 were not analyzed.	18

Table 9.  Recovery of Radioactivity in VOC Trap 2 from Group C.	19

Table 9 (contd).  Recovery of Radioactivity in VOC Trap 2 from Group C. 20

Table 10.  Recovery of Radioactivity in Nose Only Tube Rinse from Group C.	21

Table 11.  Recovery of Radioactivity in Cage Rinse from Group C.	21

Table 14.  Recovery of Radioactivity in Carcass Digest from Group C.	22

Table 15.  Total Recovery of Radioactivity in Samples from Group C.	23

Table 15 (contd).  Total Recovery of Radioactivity in Samples from Group C.	24

Table 15 (contd).  Total Recovery of Radioactivity in Samples from Group C.	25

Table 15 (contd).  Total Recovery of Radioactivity in Samples from Group C.	26

Table 15 (contd).  Total Recovery of Radioactivity in Samples from Group C.	27

Table 1.	  Male and Female Fischer 344 Rats Used on the Study	1716

Table 2.	  Exposure Conditions and Concentration of DIPE, Study A.	1817

Table 3.	  Exposure Conditions and Concentration of [2-14C]DIPE/DIPE, Study B.	1918

Table 4.	  Exposure Conditions and Concentration of [2-14C]DIPE/13CDIPE, Study C.	2019

Table 5.	  Concentration of DIPE in blood from male and female rats exposed to 3600 ppm DIPE.	2120

Table 6.	  Concentration of Isopropanol in blood from male and female rats exposed to 3600 ppm DIPE.	2221

Table 7.	  Concentration of Acetone in blood from male and female rats exposed to 3600 ppm DIPE.	2322

Table 8.	  Pharmacokinetic Analysis of DIPE, Isopropanol and Acetone in male and female rats exposed to 3600 ppm DIPE.	2423

Table 9.	  Total Recoverya of Radioactivity following Exposure to 3600 ppm [2-14C]DIPE/DIPE	2524

Table 10.	  Recovery of Radioactivity in Carcass following Exposure to 3600 ppm [2-14C]DIPE/DIPE (Group 2)	2625

Table 11.	  Recovery of Radioactivity in Excreta and Carcass following Exposure to 3600 ppm [2-14C]DIPE/13C6DIPE (Group 3)	2726

Table 12.	  Recovery of Radioactivity in Urine following Exposure to 3600 ppm [2-14C]DIPE/13C6DIPE (Group 3)	2827

Table 13.	  Recovery of Radioactivity in Feces following Exposure to 3600 ppm [2-14C]DIPE/13C6DIPE (Group 3)	2928

Table 14.	  Recovery of Radioactivity in CO2 Trap 1  following exposure to 3600 ppm [2-14C]DIPE/13C6DIPE (Group 3)	3029

Table 15.	  Recovery of Radioactivity in CO2 Trap 2 following exposure to 3600 ppm [2-14C]DIPE/13C6DIPE (Group 3)	3130

Table 16.	  Recovery of Radioactivity in Exhaled VOC Trap 1 following exposure to 3600 ppm [2-14C]DIPE/13C6DIPE (Group 3)	3231

Table 17.	  Recovery of Radioactivity in Exhaled VOC Trap 2 following Exposure to 3600 ppm [2-14C]DIPE/13C6DIPE (Group 3)	3332

Table 18.	  Cumulative Recovery of Radioactivity in Excreta and Carcass following Exposure to 3600 ppm [2-14C]DIPE/13C6DIPE (Group 3)	3433

Table 19.	  Recovery of Radioactivity in Tissues from Rat CM-02 following Exposure to 3600 ppm [2-14C]DIPE/13C6DIPE (Group 3)	3534

List of Figures

Page

  TOC \h \z \t "Figure Title" \c "Figure"   HYPERLINK \l "_Toc351999983" Figure 1.  Headspace GC-MS analysis of DIPE, acetone and isopropanol in blood.	  PAGEREF _Toc351999983 \h  373635

 HYPERLINK \l "_Toc351999985" Figure 2.  Concentration of DIPE in male and female rat blood during and following exposure to 3600 ppm DIPE for 6 hours.	  PAGEREF _Toc351999985 \h  383736

 HYPERLINK \l "_Toc351999986" Figure 3.  Concentration of Isopropanol in male and female rat blood during and  following exposure to 3600 ppm DIPE for 6 hours.	  PAGEREF _Toc351999986 \h  393837

  HYPERLINK \l "_Toc351999987"  Figure 4.  Concentration of Acetone  in male and female blood during and following exposure to 3600 ppm DIPE for 6 hours.	  PAGEREF _Toc351999987 \h  4139

  HYPERLINK \l "_Toc351999988"  Figure 5.  Noncompartmental analysis of DIPE in male (top) and female (bottom) rat blood during and following exposure to 3600 ppm DIPE for 6 hours.	  PAGEREF _Toc351999988 \h  4240

  HYPERLINK \l "_Toc351999989"  Figure 6.  Noncompartmental analysis of isopropanol in male (top) and female (bottom) rat blood during and following exposure to 3600 ppm DIPE for 6 hours.	  PAGEREF _Toc351999989 \h  4341

  HYPERLINK \l "_Toc351999990"  Figure 7.  Noncompartmental analysis of acetone in male (top) and female (bottom) rat blood during and following exposure to 3600 ppm DIPE for 6 hours.	  PAGEREF _Toc351999990 \h  4442

  HYPERLINK \l "_Toc351999991"  Figure 8.  Cumulative Excretion of Radioactivity in Urine and Feces, CO2 and Exhaled VOC Traps.	  PAGEREF _Toc351999991 \h  4543

List of Appendices

Appendix A:	Approved Study Protocol, Amendments, and Deviations

Appendix B:	Test Chemical Analysis Report

Appendix C:	Test Chemical Analysis Report:  2-14C Diisopropyl ether

		Test Chemical Analysis Report:  13C6-Diisopropyl ether

		Analysis of Reference Substances: Acetone and Isopropanol

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

Appendix E:	Prestudy Inhalation Report

Appendix F:	Inhalation Reports

Appendix G	Equations Used inExample Calculations

Appendix H 	Raw Data Tables for Radioactivity

Appendix I	Blood Sample Collection and Analysis for DIPE, Acetone and IsopropanolRaw Data Tables for DIPE, Acetone and Isopropanol in Blood

Appendix J	Pharmacokinetic Analysis

1.0  	Introduction

The purpose of this pilot study was to develop and validate procedures to be used to evaluate the adsorption, distribution, metabolism, excretion and pharmacokinetics of diisopropyl ether (DIPE) in rats.  DIPE is used as an oxygenate additive to gasoline.

2.0  	Objectives and Protocol

The objectives of this pilot study were to:

Study A

1) Collect blood from unexposed (control) male rats (n=10).

2) Develop a gas chromatography (GC) method for the analysis of the concentration of DIPE, isopropanol, and acetone in blood.

3) Develop a nose-only exposure system for exposure of male and female rats to DIPE, and conduct exposure of cannulated rats to unlabelled DIPE (n=6; 3 backup unexposed).

4) Collect blood from rats exposed to unlabelled DIPE, and measure the concentration of DIPE, isopropanol, and acetone in blood.

Study B

5) Conduct a nose-only exposure of male rats to 14C DIPE/DIPE (n=4; 1 backup unexposed).

6) Analyze the amount of 14C in whole body digests from rats exposed to 14C DIPE/DIPE.

Study C

7) Conduct a nose-only exposure of male rats to 14C DIPE/(U-13C6) DIPE (n=4; 2 backup unexposed).

8) Collect excreta from rats exposed to 14C DIPE/(U-13C6)  DIPE.

9) Analyze 14C in whole-body digests, urine, feces, exhaled CO2, and exhaled volatiles in rats exposed to 14C DIPE/(U-13C6)  DIPE.

10) Develop a method for analysis of metabolites in urine using HPLC and NMR.

Copies of the approved protocol and protocol amendments for these studies are included in Appendix A.  It should be noted that some of these objectives listed above were involved method development (development of a GC method for analysis, development of a nose only inhalation exposure system) and were not conducted according to Good Laboratory Practice.

3.0  	Materials and Methods

3.1  	Test Substance

Name:  Diisopropyl ether (DIPE; CAS No. 108-20-3)

MOLECULAR FORMULA:  C6H14O

MOLECULAR WEIGHT:  102.18

STRUCTURE:

3.1.1  	Nonradiolabeled diisopropyl ether

SOURCE OF NON-LABELED TEST SUBSTANCE:  The non-labeled DIPE was purchased from Sigma-Aldrich, Milwaukee, WI.  A certificate of analysis from the vendor indicated purity of 99.6% by GLC, with 6.6 PPM BHT

PRODUCT NUMBER:  398276

LOT NUMBER:  03658JC

The unlabeled test material was characterized at RTI by gas chromatographic (GC) analysis for purity and stability, and for identify by 1H and 13C Nuclear Magnetic Resonance (NMR) spectroscopy and by GC-mass spectroscopy.  Details of the analysis are included in Appendix B.

3.1.2  	(U-13C6)-labeled diisopropyl ether

SOURCE OF 13C-LABELED TEST SUBSTANCE:  (U-13C6) Substituted DIPE was obtained from ISOTEC, Miamisburg, OH.  A certificate of analysis from the vendor indicated purity of 95.9%.

PRODUCT NUMBERS:  ISOTEC Number T83-03014

				ALDRICH Number 632384

LOT NUMBER:  ST1187

The (U-13C6)-labeled diisopropyl ether was characterized at RTI by gas chromatography-mass spectrometry analysis for identity, and by GC-FID with a purity of 94.9%.

3.1.2  	[2-14C]-labeled diisopropyl ether

Position of radiolabel:	[2-14C]-

Source:	American Radiolabeled Chemicals, Inc. St. Louis MO, prepared as a custom synthesis

Specific Activity:	2.1 mCi/mmol

Product No.:	ARC-3099

Lot No.:	070626

Identity:	The identity of [2-14C]-diisopropyl ether was confirmed by chromatographic comparison with unlabeled DIPE using HPLC coupled with radioactivity detection.

Purity:	The radiochemical purity of [2-14C]-diisopropyl ether was determined chromatographically to be 97.23 %, using the HPLC system described in Appendix C.

The labeled test material was characterized at RTI by HPLC coupled with radioactivity detection for purity and stability, and by HPLC coupled with radioactivity detection and refractive index detection.  Details of the analysis are included in Appendix C.

3.2  	Reference Substances

Isopropanol.   2-propanol, product number 34863, lot number 05761JC was obtained from Sigma-Aldrich.  The purity specified by the Vendor’s certificate of analysis was 99.98%.  This material was assigned a chemical receipt number of DPK-B-0005.  Identity of the material was verified by 1H and 13C NMR spectroscopy, and by gas chromatography coupled with mass spectrometry.

Acetone, product number 34850, lot number 53250 was obtained from Sigma-Aldrich.  Purity from the Vendor Certificate of Analysis was 99.9%. This material was assigned a chemical receipt number of DPK-B-004.  Identity of the material was verified by 1H and 13C NMR spectroscopy, and by gas chromatography coupled with mass spectrometry.

3.3  	Test System

Source:  Male 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.

Diet:  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 60(70 (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 before and after the study for analysis of diisopropyl ethertertiary butanol, 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.   All of the analytes were below the reporting limit for the method in the samples obtained before and after the study (tertiary butanol 1 µg/L, tertiary amyl methyl ether 0.1 µg/L, ethyl tertiary butyl ether 0.1 µg/L, diisopropyl ether 0.1 µg/L, and methyl tertiary butyl ether 0.1 µg/L).

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).  Cannulated rats used in Study A were housed individually in cages with dimensions of 9 ¼" x 10.5" x 8 ¼" high (approximately 76.3 sq. in. floor space)

Following exposure in Study C, 4 male rats were housed in individual glass metabolism chambers for 7 days for collection of urine, feces, exhaled CO2, and exhaled volatiles.  Two unexposed male rats were housed in individual glass metabolism chambers for 2 days for collection of urine, feces, exhaled CO2, and exhaled volatiles for determination of radioactivity background.

Quarantine:  Rats were quarantined for a minimum of one week before use on a study, with the exception of cannulated rats which were quarantined for 1-2 days.  A veterinarian or qualified designee examined the animals prior to their release from quarantine.

Randomization:  The ear tag numbers of animals were assigned in numerical order to sequential values using a series of computer generated numbers as described in SOP DPK-HUS-001 Assignment of Animals into Groups, using the procedure for Assignment of Animals within a Single Group or Multiple Groups without Regard to Weight Mean and Range.  Four groups were prepared:  Study A (n=9 for males and females), Study B (n=4), Study C (n=4).

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 64(79 (F (18 (C - 26 (C) for temperature and 30(70% 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 exposure to unlabeled DIPE (Study A), 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 [2-14C]DIPE/DIPE (Study B), 14C 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 [2-14C]DIPE/(U-13C6)DIPE (Study C), 14C DIPE was added to an appropriate amount of (U-13C6)DIPE for generation of the exposure atmosphere.   The amount of labeled and (U-13C6)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 [2-14C]DIPE/DIPE, [2-14C]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 nominal specific activity of the DIPE mixture was then calculated.  This was verified by weighing an aliquot into a sealed flask, recording the weight added, and adding solvent, and recording the weight added.  Aliquots of the solution of DIPE were placed in scintillation vials, and the weight added was recorded.  The amount of solution added was calculated based on the density of the solvent.  Ultima Gold™ scintillation cocktail (Perkin Elmer) was added to the scintillation vials, and the amount of radioactivity added was determined by liquid scintillation spectroscopy (LSS).  The specific activity of the labeled DIPE was then calculated from the data obtained.

A procedure analogous to that described above was followed for the preparation of [2-14C]DIPE/(U-13C6)DIPE.

The exposure atmosphere concentration was monitored using a calibrated Miran IR detector.   The concentration of DIPE was monitored continuously at the inlet to the tower, and at the outlet.  The stability of DIPE under the conditions of administration was monitored by sampling the inlet of the exposure tower during the exposure and at the end of the exposure (Appendix F).  Samples of the exposure atmosphere were collected using a 1ml syringe, and bubbled through water for analysis by HPLC, and through methanol for analysis by GC-MS.

3.5  	Inhalation Exposure

A single nose-only inhalation exposure to [2-14C]DIPE/DIPE was conducted with a total of 9 8 male and 9 8 female rats for Study A on September 27, 2007.  A single nose-only inhalation exposure to [2-14C]DIPE/DIPE was conducted with a total of 4 male rats for Study B on October 3, 2007.   A single nose-only inhalation exposure to [2-14C]DIPE/(U-13C6)DIPE was conducted with a total of 4 male rats for Study C on October 4, 2007. For each study, the DIPE preparation was drawn into a clean syringe.  The generation system consisted of a syringe containing DIPE with a syringe pump to deliver the chemical to the air supply of the exposure chamber.  The DIPE exposure atmosphere was generated by pumping liquid DIPE into the air stream flowing into a Cannon nose-only exposure system.  The syringe pump and clean air flow rate were set such that a target concentration of 3600 ppm was achieved.  The exposure air flow rate delivered to each animal was at least 1.5 times the animal minute ventilation rate.  The air supply was controlled with an electronic mass flow meter to maintain a total air flow of at least 12 air changes per hour.  The generation and delivery system were placed in a chemical hood to contain any DIPE that might leak from the system.

The exposure system (see Appendix E and Appendix F) consisted of a Cannon flow-past nose-only exposure system containing 52 exposure ports.  This chamber is a dynamic non-rebreathing system that allows for the simultaneous exposure of up to 51 animals.  The chamber is cylindrical in shape, and is constructed in stainless steel.  Unused ports were plugged with solid metal rods to reduce consumption of the exposure material.  The Cannon nose-only chamber operated on a push-pull basis.  The sampling lines and main exposure atmosphere delivery lines were Teflon.   The incoming air for the exposure system was filtered to eliminate the possibility of contamination in the air supply.  To the extent possible, tThe air supply temperature and relative humidity were maintained between 64 to 79 degrees Fahrenheit and 30 to 70 %, respectively.  The chamber exhaust flow was adjusted to maintain a slight negative pressure during the exposure to prevent DIPE from entering the laboratory area. The chamber exhaust was filtered through a disposable charcoal filter and disposed at the end of the exposure.   Closed nose-only tubes were used to hold the test animals during inhalation exposures.  The inhalation system was strategically placed in a chemical hood to prevent any DIPE from entering the laboratory.

3.5.1 Exposure Analysis

Prior to the inhalation exposure of rats, the performance of the inhalation system was verified to ensure that the test atmosphere could be generated on the nose only tower to achieve the test concentration over the duration of the exposure (≤ 10% difference between actual and target concentration), and that the port to port variability was within accepted limits (≤ 10% difference in measurement taken at multiple locations on the tower). A report describing the preexposure characterization of the inhalation system is attached to this report as Appendix E, and a report reports characterizing the exposures of rats is are attached as Appendix F.  Note that during the sampling of air at the ports on the inhalation exposure system to establish port to port variability, the air sampled was diluted with additional makeup air added to enable more rapid sampling.  The sampling was conducted to demonstrate port to port variability, and with the same proportion of makeup air added over the course of the exposure, the determination of variability is valid, even though the mean concentration sampled at the port is less than the 3600 ppm concentration determined at the inlet.

3.6  	Collection of Samples

3.6.1	Blood Sample Collection

For Study A, blood samples were collected by jugular vein cannula prior to dosing from each rat (time = 0), and from three rats of each sex, at approximately 5 min, 10 min, 15 min, 30 min, and at approximately 1, 2, 4 and 6 hr after the exposure initiation.  At the end of the 6 hr exposure, all rats were removed from the inhalation exposure tower, and placed in cages.  Blood was drawn from three rats at approximately 375, 390, 420, 440, 480, 600, 960, and 1440 min after the beginning of the exposure (at approximately 15, 30, 60, 80 min, and 2 hr, 4 hr, 10 hr, and 18 hr after the end of the exposure).  To facilitate the withdrawal of blood samples, animals were stagger-started on the exposure tower. At each time point approximately 100 L of blood was withdrawn using a heparinized syringe.

Blood samples 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 amount of blood was approximately 100 µl.  The vial was immediately crimped and weighed, and the weight of the blood aliquot was determined.  After the last sample has been collected from each animal, the rats were euthanized by exposure to CO2.  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 method development.  Blood samples were collected by cardiac puncture, under CO2 anesthesia, and the rats were euthanized.

3.76.12	Excreta

In Study C, 4 rats (CM-01, CM-02, CM-03, and CM-04) were placed in all-glass metabolism cages for the separate collection of urine, feces, exhaled volatiles, and exhaled CO2.  Urine was collected over dry ice at 0(8, 8(24, 24(48, 48(72, 72(96, 96(120, 120(144, and 144(168 hours after termination of exposure.  Feces were collected over dry ice at 0(24, 24(48, 48(72, 72(96, 96(120, 120(144, and 144(168 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) at 0(1, 1(3, 3(6, 6(24, 24(48, 48(72, 72(96, 96(120, 120(144, and 144(168 hours after termination of exposure.   Expired 14CO2 was collected in 1.0 N KOH at 0(1, 1(3, 3(6, 6(24, 24(48, 48(72, 72(96, 96(120, 120(144, and 144(168 hours after termination of exposure.   At the end of excreta collection, the cages were rinsed with water and with ethanol, and combined.   The rinses were analyzed for total radioactivity.  The weight of urine and/or feces collected for each sample interval was measured.  Urine and feces were analyzed for total radioactivity.  Excreta not assayed within a day of collection were stored at approximately -20 (C in the dark.

Two unexposed rats were housed in all-glass metabolism cages for 48 hours, as described above, for the collection of urine, feces, and exhaled volatiles, and exhaled CO2.  The samples collected were used to supply background samples for the analysis of radioactivity.

3.6.23	Carcass

For Study B, the amount of 14C retained was determined by placing each rat in the nose-only restraint tube in a Tedlar gas bag.  The bag was sealed, and CO2 was pumped into the bag to euthanize the rat.  The gas from the gas bag was then forced through a charcoal filter trap, to determine the amount of exhaled 14C, and the carcass was digested with 2N ethanolic NaOH.  After sample digestion, the amount of radioactivity in the carcass was determined by LSS.  Any feces in the nose-only restraint tube were collected and the amount of radioactivity in the feces sample was determined by LSS.  The nose-only tube was rinsed with water, and the amount of 14C contained in the rinse was determined by LSS.

For Studies B and C, the carcasses were digested with 2N ethanolic NaOH.  After sample digestion, the amount of radioactivity in the carcass was determined by LSS.

3.6.24	Tissues

For Study C, tissues were obtained following sacrifice from a single rat (CM-02):

 femur					cecum + large intestine + rectum

skin (hair shaved)			small intestine

subcutaneous fat			liver

abdominal fat				muscle (gastrocnemius)

brain					heart

testes (epididymis removed)		kidneys

stomach contents 			small intestine contents

stomach				large intestine + cecum contents

lungs					spleen

residual carcass

The harvested tissues were weighed and analyzed for radiochemical (14C) content by LSS.  The remaining carcass of the animal from which the tissues listed above have been taken will bewas analyzed for total radioactivity.  Tissues will bewere prepared as described in section 83.68.23 below.

3.8  7  	Analysis of Blood Samples for DIPE, Isopropanol, and Acetone

A GC-MS method was developed for quantitating DIPE, isopropanol and acetone using blood from control male Fischer 344 rats.  The methods development involved the generation of a standard curve for analysis of DIPE, isopropanol and acetone in the headspace of airtight vials containing a known volume of control blood and a known concentration of each analyte.  The stability of DIPE, isopropanol and acetone was evaluated under storage conditions that were used in this study during this method development.  This included analysis of DIPE, isopropanol and acetone immediately after spiking in control blood, after storage at room temperature (8 hr), or at approximately 4oC (24 16 hr).  The concentration of DIPE, isopropanol and acetone in the headspace of vials containing a known volume of blood from rats administered DIPE by inhalation was determined by comparison to the standard curve.  A detailed description of the analytical method is contained in Appendix C.

The method was applied to the analysis of samples obtained from jugular vein cannulated rats exposed to DIPE by inhalation.  Blood samples were obtained from cannulas (or in the cases where samples could not be obtained via the cannula, from the tail vein) as described in section 3.6.1.  Samples were transferred immediately to headspace vials, crimp sealed, and analyzed as described in Appendix C.  Vials and caps were weighed prior to addition of blood, and after addition of blood, and the weight of blood added was calculated.  Internal standard solution (10 µL) containing MTBE (50 µg/mL) and 1-propanol (500 µg/mL) was added using a 10-µL microsyringe.  After analysis by headspace GC-MS, the concentration of each analyte was corrected for the weight of blood collected.  The method is described in detail in Appendix C.

3.8  	Analysis of Samples for Total Radioactivity

Ultima Gold™ scintillation cocktail was used in all determinations of radiochemical content.  Radioactivity was determined using a Packard 1900CA Liquid Scintillation Counter.  Weights of samples collected were recorded, and aliquots were prepared by weight for scintillation counting.

3.8.1	Excreta

Duplicate aliquots of urine (approximately 0.05 g) 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™ (about 2 ml per sample), scintillation cocktail was added to the vials, and the samples were analyzed for total radioactivity by LSS.  Samples were bleached (by adding approximately 125 µl of 70% perchloric acid, and then approximately 0.3 ml of 30% H2O2) prior to addition of scintillation cocktail.  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 were analyzed for total radioactivity following solubilization in 2N ethanolic NaOH.  Duplicate samples of the solubilized carcass were analyzed.  Tissues were analyzed for total radioactivity following solubilization in Soluene-350™ (about 2 mL per tissue sample).    Liver was homogenized, and duplicate aliquots of the homogenate solubilized.  Other tissues were solubilized in their entirety or after being divided into multiple pieces.  Solubilized tissue samples were bleached (by adding approximately 125 µl 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

Aliquots of 1.0 N KOH from the exhaled breath trap for CO2 were analyzed by LSS after addition of scintillation cocktail.

3.8.4  Exhaled Volatiles Traps

Each charcoal trap was extracted by elution with dimethylformamide (DMF) to determine absorbed radioactivity.  For a single extraction, charcoal was transferred to a centrifuge tube and weighed.  DMF (8.0 ml for samples of VOC Trap 1 from 0 – 24 h, and 4.0 ml for samples collected following 24 h) was added, and the sample weighed.  After vortexing for approximately 30 seconds, the samples were centrifuged, and the supernatant was removed.  The DMF was removed following centrifugation and radioactivity was determined in duplicate aliquots of the DMF extract by LSS.

3.9  	Data Collection and Reporting

Study data was collected and reported in the Debra™ system version <IP_ADDRESS>75.  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™ 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 reported as 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.

4.0  	Results

4.1  	Analysis of Radiolabeled Test Chemical 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 99.38 ( 0.16 %.  Following completion of the study, test chemical purity was determined to be 99.71 63 ( 0.01 006 %.  The radiochemical purity of 2-14C DIPE specified by the vendor was 96.83 % by HPLC, and was verified by HPLC at RTI prior to the inhalation exposure of rats as 9897.37 23 ( 0.2404% (Appendix C).  A sample of the exposure atmosphere was taken from the tower inlet to measure purity of the radiolabeled test chemical during the exposure.  The purity of the radiolabeled material was determined to be 94.6 ( 2.4 %.  The purity of the (U-13C6) DIPE specified by the vendor was 95.9 % and was verified by NMR spectrocscopyspectroscopy, and by GC as 95.17 ± 0.03 %.  Analysis of the reference substances acetone and isopropanol is reported in Appendix DC.

4.2  	Test Chemical Formulation

For Study A, unlabeled DIPE was used for generating the exposure atmosphere.  For Study B, the test chemical was prepared by mixing unlabeled DIPE with 2-14C labeled DIPE, and used without further dilution.  The specific activity of the formulation is reported in Table 23, and was measured as 0.4503 µCi/mg.   For Study C, the test chemical was prepared by mixing (U-13C6) DIPE with 2-14C labeled DIPE, and used without further dilution.  The specific activity of the formulation is reported in Table 34, and was measured as 0.4241 µCi/mg.ch

4.3  	Inhalation exposure

The body weights of the animals used during the course of Studies A, B and C are shown in Table 1.  The exposure concentration data for each six-hour exposure of rats to a target concentration of 3600 ppm DIPE is shown in Table 2, 3, and 4.  The details of the inhalation exposure system are described in 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 F, Inhalation Summary Reports: DIPE, 14C-DIPE/DIPE and 14C-DIPE/13C-DIPE Nose-Only Inhalation Exposure at RTI International.  The actual exposure concentration was 3643 ( 177 ppm for Study A (Table 2), 3529 ( 40 ppm for Study B (Table 3), 3334 ( 99 ppm for Study C  (Table 4).   The exposure duration for Study C was less than 6 hours, with the limited supply of 13C DIPE providing an exposure duration of 5 hours and 40 minutes.  Analyses of samples of the exposure atmosphere are reported in Appendix F, Analysis of Exposure Atmospheres: Diisopropyl Ether.   GC-MS analysis of samples of the exposure atmosphere sampled during Studies A, B and C confirmed the presence of DIPE or 13C6-DIPE in the exposure atmosphere near the beginning and end of the exposures, verifying the stability of DIPE in the inhalation system. With the exception of one sample that showed a small peak containing acetonitrile, that is thoguththought to have arisen from contamination of a sampling syringe, there were no significant impurity peaks suggesting that the DIPE is stable in the inhalation system.  Radioactivity from air samples collected during the course of the inhalation exposures for Studies B and C chromatographed with the expected retention time of DIPE on HPLC.  However, the levels of radioactivity recovered were insufficient to reliably establish purity.

4.4  	DIPE, Acetone and Isopropanol in Blood During and  Following Exposure

DIPE, isopropanol and acetone were determined by headspace GC-MS analysis in blood samples obtained from jugular vein cannulated male and female rats (Study A, Group 1, rats AM-01 – AM-08, AF-01 – AF-08).  The concentrations determined are presented in Tables 5 - 7 9 and in Figures 1 - 34.  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 16 hr. In the male rats, blood concentrations of isopropanol was were below LOQ in preexposure samples, all three samples collected at 5 min, and in one of the three collected at 10 min.  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 16 hr.  In the female rats, isopropanol blood concentrations was were below LOQ in preexposure samples, and all samples collected at 5 10, and 15 min.  The concentration of isopropanol rose steadily during the exposure, and then declined gradually.  In male rats, blood concentrations of isopropanol peaked at approximately twice those of female rats.  All samples were below LOQ by 16 hr.  In male and female rats, acetone was above LOQ in 5 of the 6 preexposure samples.  Acetone levels in both male and female rats rose steadily during the exposure, and in males continued to rise following exposure until 8 hr following the initiation of exposure.  In both males and females, the acetone levels were approximately the same between 6 and 8 hours following the exposure, and then declined rapidly reaching preexposure levels by 16 hrs in females and 24 hrs in males.  The concentration achieved in males exceeded the range of the calibration curve, and for some samples obtained between 6 4 hours and 10 hours, concentrations 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.   Male rats reached preexposure levels by 24 hours.

Noncompartmental pharmacokinetic analysis was conducted using the mean concentration of DIPE, isopropanol or acetone rather than individual values.  With the alternating sampling of the animals during and following exposure, the use of mean values with all of the data points provides the best pharmacokinetic analysis.   With noncompartmental analysis, the elimination rate constant and half life are determined from the rate of decline of blood concentration following the end of the exposure.  Semi-log plots of concentration vs. time for DIPE in blood are shown in Figure 45.   Pharmacokinetic analysis of DIPE indicated a half life of 0.92 h in male rats, and 0.76 h in female rats (Table 8).  Isopropanol had an apparent half life of 1.16 and 1.23 h in male and female rats, respectively (Table 8 and Figure 56).  Acetone had an apparent half life of 2.31 and 2.32 h in male and female rats, respectively (Table 8 and Figure 67).  Acetone achieved the highest levels in blood (Cmax values of 319 and 211 µg /mL in male and female rats, respectively), compared with DIPE (Cmax values of 56 and 66 µg /mL in male and female rats, respectively), and isopropanol (Cmax values of 66 and 36 µg /mL in male and female rats, respectively). The peak concentrations of acetone achieved in male rats were approximately 50% higher than those in female rats, and for isopropanol the peak concentrations in males were almost twice those in females.    Tmax values for DIPE were at 4 h in male rats and 6 h in female rats.  The Tmax values for acetone and isopropanol occurred at between 6 and 6.5 h, suggesting that the metabolism of DIPE to isopropanol and acetone occurs rapidelyrapidly.   Acetone achieved the highest area under the curve (AUCall values of 3208 and 1735 µg·hr/mL in male and female rats, respectively) compared with isopropanol (AUCall values of 339 and 156 µg·hr/mL in male and female rats, respectively) and DIPE (AUCall values of 312 and 363 µg·hr/mL in male and female rats, respectively).

4.5  	Radioactivity Retained Following Exposure

Four rats were euthanized immediately following inhalation exposure to [2-14C]DIPE/DIPE (Study B, Group 1, rats BM-01, BM-02, BM-03, and BM-04).  The amount of radioactivity retained in the carcass was determined by solubilization followed by scintillation counting.  The total radioactivity retained and the estimated dose in mg/kg are presented in Table 9.  The amount of radioactivity retained in Group B ranged from approximately 33 to 34 35 µCi.  The estimated dose was 373 ( 8 mg/kg.   This estimate of dose included radioactivity recovered in carcass, a VOC trap that was used to trap any exhaled volatiles while the rat was in the transfer bag, the nose only tube wash, and feces recovered from the nose only tube.   The vast majority (98.6%) of the radioactivity was recovered in the carcass (Table 10).

4.6  	Excretion of Radioactivity

Four rats were placed in metabolism cages for collection of urine and feces and exhaled breath immediately following the inhalation exposure to [2-14C]DIPE/DIPE (Study C, Group 3, Rats CM-01, CM-02, CM-03, and CM-04).  Transfer of the rats from the inhalation tower to the metabolism cage was conducted with the rat retained in the nose-only tube contained within a polyethylene bag.  Following transfer of the rat to the metabolism cage, radioactivity retained in the bag was recovered by washing with water.  The nose-only tube was washed and any feces were collected for digestion and scintillation counting.  The rats were maintained in the metabolism cages for 7 days following the end of the exposure.  The exhaled breath traps from one rat were not analyzed, but were saved to provide an opportunity for analysis at a later date, and are not included in calculations of recovery.  The total recovery of radioactivity from all of the samples collected from rats in Group 3, Study C is presented in Table 9, and ranged from approximately 25 to 28 µCi (with the exception of rat CM-04,where exhaled breath traps were saved for possible future analysis).  The estimated dose was 299 318 ( 7 8 mg/kg (excluding CM-04, Table 9).

The amount of radioactivity in urine, feces, exhaled VOCs, and CO2 expressed as a percentage of the total radioactivity recovered is presented in Table 11.  Exhalation of volatile material was the primary route of elimination, with 71% of the recovered radioactivity trapped in the Exhaled VOC Traps. Exhaled 14CO2 accounted for approximately 20.2 % of the recovered radioactivity.  Approximately 5 % and 1% were excreted in urine and feces, respectively.

Radioactivity in urine was excreted primarily in the first 24 hours (Table 12), with 3% and 1.5% at 12 8 and 24 hours, respectively.  Less than or equal to 0.2 % or less of the radioactivity was recovered in urine at 48, 72, 96, 120, 144, and 168 hours.  In feces (Table 13), approximately 0.6 % of the recovered activity was found in the 0-24 h samples, which contained the largest amount of fecal radioactivity.

Radioactivity in the first CO2 trap was excreted primarily in the first 24 hours (Table 14).  Little radioactivity was recovered in the second CO2 trap (Table 15).  Many of the samples collected 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 the first 24 hours (Table 16).  Each of the Exhaled VOC Trap 1 samples collected between 0 and 24 hours contained a substantial portion of the dose.  Very little of the dose was trapped in the VOC Trap 2 samples (Table 17), with the exception of the 6-24 h samples for rats CM-01 and CM-02.

The cumulative excretion of radioactivity (Table 18) shows that approximately 94% of the radioactivity was recovered within 24 h following exposure, and 95% of the activity was recovered by 48 hours following exposure.

Tissue recovery was evaluated for one rat at 7 days following exposure (Table 19).  Liver contained approximately 0.12 % of the recovered radioactivity.  All other tissues collected contained lower percentages of the dose.

5.0  	Discussion

The objectives of the study were accomplished.  An inhalation exposure system was developed, and used to conduct the exposures to 3600 ppm DIPE.   A headspace GC-MS method for analysis of DIPE, isopropanol, and acetone was developed, and applied to samples from male and female rats exposed to DIPE.  The disposition of 14C DIPE was evaluated in male rats, and determined immediately after exposure, and up to 7 days following exposure.  The extent of excretion of metabolites in urine was less than 5% of the dose, so the objective of determining the identity of metabolites in urine was modified.

 Comparison of the exposure of rats to 3600 ppm [2-14C]DIPE/DIPE euthanized immediately following exposure (372 ( 8 mg/kg) with that of rats euthanized following 7 days of collection of excreta and exhaled breath (299 318 ( 7 8 mg/kg) yielded values that were very similar.  Normalization of these values based on the actual exposure concentrations and the duration of exposure (Table 20) reduced the extent of the difference, with 0.176 mg/kg/ppm.hr immediately after exposure, and 0.168 mg/kg/ppm.hr at 7 days, indicating a recovery of approximately 95.6%.  This suggested that there were no significant losses of radioactivity from the various sample types during processing of radioactivity.

Radioactivity was rapidly excreted by exhalation as volatile components (71% of the recovered radioactivity) and as 14CO2 (2520.2% of the recovered radioactivity).  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 02.6% was recovered in the carcass.

DIPE in blood was readily detected during and after exposure.  DIPE levels fell rapidly following the end of the exposure, reaching the limit of quantitation by 10 hours after initiation of exposure.   Acetone and isopropanol were detected in blood, increasing with the duration of exposure and decreasing following the end of the exposure.  The acetone levels were substantially higher than those of DIPE and isopropanol.  The conversion of DIPE to isopropanol and acetone is consistent with the high recovery of radioactivity in the exhaled breath volatiles and as 14CO2.

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:

Protocol and any amendments

Animal receipt records

Quarantine records

Temperature and humidity records for the treatment rooms

Animal research facility room logs

Feed and water analysis for contaminants

Randomization records

Test chemical receipt, storage, and use records

Balance calibration log references

Correspondences

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, under the responsibility of the RTI Quality Assurance Unit, 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 10 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: 	J.S. Scott-Emuakpor, D.V.M.

Animal Facility Supervisor:

Laboratory Staff:  	N. F. Gaudette, BS

	Y. Hong, BS

	R. W. Snyder, MS

	M. P. Gower

Secretary: 	K. G. Ancheta

Quality Assurance Reviewer:  K. Collier

	C. Ingalls

	C. Keller

	B. Rauscher

CIIT Centers for Health Research

Principal Investigator:: 	R Arden James.

Laboratory Staff:  	K. Roberts

Quality Assurance Reviewer:  P. O’Brien Pommerleau

	A. Matrone

Table   SEQ Table \* ARABIC  1 .	  Male and Female Fischer 344 Rats Used on the Study

Group	Subject	Species	Strain	Gender	Body Wta

Group 1 Study A	AM-01	Rat	Fischer 344	Male	185.10 g

Group 1 Study A	AM-02	Rat	Fischer 344	Male	172.51 g

Group 1 Study A	AM -03	Rat	Fischer 344	Male	191.92 g

Group 1 Study A	AM-04	Rat	Fischer 344	Male	185.81 g

Group 1 Study A	AM-05	Rat	Fischer 344	Male	163.70 g

Group 1 Study A	AM-06	Rat	Fischer 344	Male	173.98 g

Group 1 Study A	AM -07	Rat	Fischer 344	Male	164.56 g

Group 1 Study A	AM-08	Rat	Fischer 344	Male	177.08 g

Group 1 Study A	AF-01	Rat	Fischer 344	Female	117.23 g

Group 1 Study A	AF-02	Rat	Fischer 344	Female	122.97 g

Group 1 Study A	AF-03	Rat	Fischer 344	Female	112.56 g

Group 1 Study A	AF-04	Rat	Fischer 344	Female	114.36 g

Group 1 Study A	AF-05	Rat	Fischer 344	Female	118.86 g

Group 1 Study A	AF-06	Rat	Fischer 344	Female	119.86 g

Group 1 Study A	AF-07	Rat	Fischer 344	Female	116.93 g

Group 1 Study A	AF-08	Rat	Fischer 344	Female	112.15 g

Group 2 Study B	BM-01	Rat	Fischer 344	Male	200.58 g

Group 2 Study B	BM-02	Rat	Fischer 344	Male	203.03 g

Group 2 Study B	BM-03	Rat	Fischer 344	Male	202.66 g

Group 2 Study B	BM-04	Rat	Fischer 344	Male	197.58 g

Group 3 Study C 	CM-01	Rat	Fischer 344	Male	188.27 g

Group 3 Study C 	CM-02	Rat	Fischer 344	Male	187.41 g

Group 3 Study C 	CM-03	Rat	Fischer 344	Male	208.47 g

Group 3 Study C 	CM-04	Rat	Fischer 344	Male	190.91 g

Group 4 Study C BKGb	CB-01

Fischer 344	Male	197.34 g

Group 4 Study C BKGb	CB-02

Fischer 344	Male	200.99 g

aBody weight on the day of exposure

bAnimals used in Group 3 and Group 4 were not exposed to [2-14C]DIPE/DIPE, and provided samples for determination of background only.

Table   SEQ Table \* ARABIC  2 .	  Exposure Conditions and Concentration of DIPE, Study A.

	Target  (ppm)

Number of Open Ports	3600

17

ACC - Inlet	Mean	3643

(ppm)	Std Dev	177

	No. of Data Points	14

ACC - Exhaust	Mean	3470

(ppm)	Std Dev	48

	No. of Data Points	14

Exposure	Mean	76.3

Temperature	Std Dev	2.2

(°F)	No. of Data Points	14

Exposure	Mean	37

Relative Humidity	Std Dev	3

(%)	No. of Data Points	14

Exposure	Mean	0.0

Static Pressure	Std Dev	0.0

(in H20)	No. of Data Points	14

1ACC Analytical Chamber Concentration

Table   SEQ Table \* ARABIC  3 .	  Exposure Conditions and Concentration of [2-14C]DIPE/DIPE, Study B.

Analysis results

Treatment name: 		DIPE 3600 ppm Nose-Only Inhalation

Dpm/g of dose prep:	 	999562646.6 dpm/g

Molecular weight: 		102.18

S/A comp/dose µCi: 		0.4503 µCi/mg

Exposure Summary

	Target  (ppm)

Number of Open Ports	3600

125

ACC1 - Inlet	Daily mean	3529

(ppm)	Std Dev	40

	No. of Data Points	13

ACC - Exhaust	Daily mean	3357

(ppm)	Std Dev	130

	No. of Data Points	13

Exposure	Daily mean	72.3

Temperature	Std Dev	0.2

(°F)	No. of Data Points	13

Exposure	Daily mean	42

Relative Humidity	Std Dev	0

(%)	No. of Data Points	13

Exposure	Daily mean	-0.15

Static Pressure	Std Dev	0.0

(in H20)	No. of Data Points	13

1ACC Analytical Chamber Concentration

Table   SEQ Table \* ARABIC  4 .	  Exposure Conditions and Concentration of [2-14C]DIPE/13CDIPE, Study C.

Analysis results

Treatment name: 		DIPE 3600 ppm Nose-Only Inhalation

Dpm/g of dose prep:	 	941599268.6 dpm/g

Molecular weight: 		108.18

S/A comp/dose µCi: 		0.4241µCi/mg

Exposure Summary

	Target  (ppm)

Number of Open Ports	3600

5

ACC1 - Inlet	Daily mean	3334

(ppm)	Std Dev	99

	No. of Data Points	13

ACC - Exhaust	Daily mean	1650

(ppm)	Std Dev	113

	No. of Data Points	12

Exposure	Daily mean	72.2

Temperature	Std Dev	0.2

(°F)	No. of Data Points	12

Exposure	Daily mean	42

Relative Humidity	Std Dev	0

(%)	No. of Data Points	12

Exposure	Daily mean	-0.15

Static Pressure	Std Dev	0.03

(in H20)	No. of Data Points	12

1ACC Analytical Chamber Concentration

Table   SEQ Table \* ARABIC  5 .	  Concentration of DIPE in blood from male and female rats exposed to 3600 ppm DIPE.

	Male Rats	Female Rats

Time (hr)	DIPEa µg/mL in blood	DIPEa µg/mL in blood

	Mean	SD	Mean	SD

0.0833	19.298	5.1557	20.219	2.8453

0.1667	25.213	5.4138	29.693	4.3215

0.25	31.753	2.6903	31.596	4.0032

0.5	37.948	10.504	42.048	0.3598

1	44.554	6.6781	47.609	4.4577

2	48.719	7.9949	60.344	3.5563

4	55.708	2.4736	60.562	6.0246

6	47.557	17.696	65.61	5.5047

6.25	17.939	5.8199	18.672	1.1612

6.5	10.337	1.8071	12.058	2.5825

7	7.8988	0.6505	6.1304	0.8475

7.33	3.8405	1.3364	4.4085	0.606

8	2.2091	0.2765	1.9226	0.4641

10	0.5031	0.2859	0.387	0.0259

16	<LOQb	<LOQ	<LOQ	<LOQ

24	<LOQ	<LOQ	<LOQ	<LOQ

a Values are mean ± SD  of 3 animals.

b Less than the limit of quantitation.

Table   SEQ Table \* ARABIC  6 .	  Concentration of Isopropanol in blood from male and female rats exposed to 3600 ppm DIPE.

	Male Rats	Female Rats

Time (hr)	          Isopropanola µg/mL in blood	        Isopropanola  µg/mL in blood

	Mean	SD	Mean	SD

0.0833	<LOQb	<LOQ	<LOQ	<LOQ

0.1667	2.655	0.3136	<LOQ	<LOQ

0.25	3.3402	0.2985	<LOQ	<LOQ

0.5	5.8764	1.0548	3.3292	0.1971

1	11.665	0.6519	5.1457	0.7126

2	22.367	4.2463	10.752	0.4521

4	42.303	3.8943	21.477	5.0914

6	66.182	8.3302	31.609	1.4026

6.25	63.386	6.378	35.708	4.836

6.5	63.394	7.4217	25.59	1.9423

7	50.948	6.6648	25.368	4.4118

7.33	43.933	5.1632	15.16	1.6226

8	31.884	3.6357	11.813	2.648

10	9.1135	0.6807	3.4767	0.1157

16	<LOQ	<LOQ	<LOQ	<LOQ

24	<LOQ	<LOQ	<LOQ	<LOQ

a Values are mean ± SD  of 3 animals.

b Less than the limit of quantitation.Table   SEQ Table \* ARABIC  7 .	  Concentration of Acetone in blood from male and female rats exposed to 3600 ppm DIPE.

	Male Rats	Female Rats

Time (hr)	        Acetonea µg/mL in blood	          Acetonea µg/mL in blood

	Mean	SD	Mean	SD

0.0833	5.3986	0.9902	4.3126	2.0263

0.1667	8.6662	1.5401	5.0105	0.7406

0.25	12.545	2.4086	8.5253	1.8353

0.5	25.403	3.4346	16.779	4.5256

1	50.448	4.9173	36.046	1.9947

2	98.814	5.3419	70.829	5.0068

4	203.31	16.378	147.54	25.297

6	288.42	76.855	209.39	10.934

6.25	298.9	19.328	211.33	8.349

6.5	319.38	15.137	190.35	18.436

7	312.47	22.028	208.43	18.258

7.33	315.15	19.22	197.69	22.746

8	301.87	16.539	199.75	40.378

10	230.07	3.7206	115.47	4.5947

16	66.649	10.736	1.8999	0.0564

24	1.646	0.2375	2.7144b	n/a

a Values are mean ± SD  of 3 animals.

bOne animal, with two Two animals with measurements less than the limit of quantitation.Table   SEQ Table \* ARABIC  8 .	  Pharmacokinetic Analysis of DIPE, Isopropanol and Acetone in male and female rats exposed to 3600 ppm DIPE.

	Units	DIPE	DIPE	Isopropanol	Isopropanol	Acetone	Acetone

Male	Female	Male	Female	Male	Female

Cmax	µg /mL	55.7082	65.6099	66.1823	35.7084	319.3774	211.3721

Tmax	hr	4	6	6	6.25	6.5	6.25

Half Life	hr	0.9165	0.7587	1.1598	1.2261	2.3107	2.3195

Lambda z	1/hr	0.7563	0.9136	0.5653	0.5976	0.3	0.2988

AUCall	µg·hr/mL	311.6541	363.2418	338.858	156.0429	3208.097	1734.658

Table   SEQ Table \* ARABIC  9 .	  Total Recoverya of Radioactivity following Exposure to 3600 ppm [2-14C]DIPE/DIPE

Rat	Total DPM Recovered	Total (Ci	Dose (mg)	Bodyweight (g)	Dose (mg/kg) Mean Dose (mg/kg) c	SD d

BM-01	77199927	34.747	77.23	200.58	385.1	372.99	8.33

BM-02	74997744	33.7828	75.03	203.03	369.6

BM-03	74152179	33.4019	74.18	202.66	366.1

BM-04	73329600	33.0314	73.36	197.58	371.3

CM-01	55226372	24.8767	58.6555.25	188.27	311.5293.5	317.89 299.45  7.677.23

CM-02	57600304	25.9461	61.6757.63	187.41	326.4307.5

CM-03	61974740	27.9165	65.8262.00	208.47	315.7297.4

CM-04b	15313501	6.8980	16.2615.32	190.91	85.280.2

a Total recovery calculated from the total DPM recovered in all samples

b Total recovery for CM-04 does not include the analysis of exhaled breath traps, which have been saved for other possible analysis.

c Mean dose mg/kg indicates the mean of 4 rats for BM and the mean of 3 rats for  CM.

d SD indicates the standard deviation of  the dose in mg/kg of 4 rats for BM and of 3 rats for  CMTable   SEQ Table \* ARABIC  10 .	  Recovery of Radioactivity in Carcass following Exposure to 3600 ppm [2-14C]DIPE/DIPE (Group 2)

	Group 2 Study B	Group 2 Study B	Group 2 Study B	Group 2 Study B	Group 2 Study B	Group 2 Study B

Sample	BM-01	BM-02	BM-03	BM-04	Mean	SD

Nose Only Tube Rinse	0.22	0.17	0.24	0.16	0.199	0.037

Carcass Digest	98.20	97.75	99.53	98.73	98.552	0.764

Exhaled VOC	0.63	1.17	0.00	0.52	0.773	0.350

Exposure Urine	N.S.	N.S.	N.S.	N.S.	0.000	0.000

Exposure Feces	0.95	0.91	0.23	0.38	0.617	0.365

Exp. Urine Sample 2	N.S.-	N.S.-	N.S.-	0.21	0.052	0.105

Total	100.00	100.00	100.00	100.00	100.000	0.000

* Value is below 3 x background for the sample.aN.S. indicates no sample.

* Value is below 3 x background for the sample.

Table   SEQ Table \* ARABIC  11 .	  Recovery of Radioactivity in Excreta and Carcass following Exposure to 3600 ppm [2-14C]DIPE/13C6DIPE (Group 3)

	Group 3 Study Ca	Group 3 Study Ca	Group 3 Study Ca	Group 3 Study Ca Group 3 Study Ca

	Male	Male	Male

Sample	CM-01	CM-02	CM-03	Mean	SD

Urine	5.5382	4.2344	4.9174	4.897	0.652

Feces	1.0493	0.8501	1.4376	1.112	0.299

CO2 Trap 1	17.5640	17.6930	19.5450	18.267	1.108

CO2 Trap 2	2.6872	1.1723	2.0266	1.962	0.760

Exhaled VOC Trap 1	64.7990	69.4660	68.0450	67.436	2.392

Exhaled VOC Trap 2	5.4380	3.8925	0.5220	3.284	2.514

Exposure Urine	N.S.	N.S.	N.S.	0.000	0.000

Exposure Feces	0.0198	N.S.	0.1172	0.046	0.063

Nose Only Tube Rinse	0.2170	0.1873	0.3494	0.251	0.086

Carcass Digest	2.6486	2.1383	3.0034	2.597	0.435

Cage Rinse	*0.0394	*0.0392	*0.0366	0.038	0.002

Total	100.00	100.00	100.00	100.000	0.000

a CM-04 is excluded from the table, since exhaled breath traps were saved for possible future analysis.

bN.S. indicates no sample.

* Value is below 3 x background for the sample.Table   SEQ Table \* ARABIC  12 .	  Recovery of Radioactivity in Urine following Exposure to 3600 ppm [2-14C]DIPE/13C6DIPE (Group 3)

Group 3 Study CaGroup 3 Study C	Group 3 Study CaGroup 3 Study C	Group 3 Study CaGroup 3 Study C	Group 3 Study CaGroup 3 Study C	Group 3 Study CaGroup 3 Study C

Male	Male	Male

CM-01	CM-02	CM-03	Mean	SD

Urine	0 - 8 h	3.0834	2.9938	2.9543	3.011	0.066

Urine	8 h - 24 h	2.0143	0.8914	1.6701	1.525	0.575

Urine	24 h - 48 h	0.2756	0.1731	0.1519	0.200	0.066

Urine	48 h - 72 h	0.0774	0.0797	0.0588	0.072	0.011

Urine	72 h - 96 h	0.0374	0.0448	0.0346	0.039	0.005

Urine	96 h - 120 h	0.0243	0.0231	0.0194	0.022	0.003

Urine	120 h - 144 h	0.0138	0.0141	0.0180	0.015	0.002

Urine	144 h - 168 h	0.0120	0.0144	0.0103	0.012	0.002

Subtotal

5.538	4.234	4.917	4.897	0.652

a CM-04 is excluded from the table, since exhaled breath traps were saved for possible future analysis.

* Value is below 3 x background for the sample.

Table   SEQ Table \* ARABIC  13 .	  Recovery of Radioactivity in Feces following Exposure to 3600 ppm [2-14C]DIPE/13C6DIPE (Group 3)

Group 3 Study CaGroup 3 Study C	Group 3 Study CaGroup 3 Study C	Group 3 Study CaGroup 3 Study C	Group 3 Study CaGroup 3 Study C	Group 3 Study CaGroup 3 Study C

Male	Male	Male

CM-01	CM-02	CM-03	Mean	SD

Feces	0 h - 24 h	0.5510	0.3695	0.8500	0.590	0.243

Feces	24 h - 48 h	0.1480	0.1845	0.2471	0.193	0.050

Feces	48 h - 72 h	0.1816	0.1262	0.0861	0.131	0.048

Feces	72 h - 96 h	0.0606	0.0490	0.1303	0.080	0.044

Feces	96 h - 120 h	0.0366	0.0521	0.0494	0.046	0.008

Feces	120 h - 144 h	0.0431	0.0343	0.0126	0.030	0.016

Feces	144 h - 168 h	0.0284	0.0345	0.0620	0.042	0.018

Subtotal

1.049	0.850	1.438	1.112	0.299

a CM-04 is excluded from the table, since exhaled breath traps were saved for possible future analysis.

* Value is below 3 x background for the sample.

Table   SEQ Table \* ARABIC  14 .	  Recovery of Radioactivity in CO2 Trap 1  following exposure to 3600 ppm [2-14C]DIPE/13C6DIPE (Group 3)

Group 3 Study CaGroup 3 Study C	Group 3 Study CaGroup 3 Study C	Group 3 Study CaGroup 3 Study C	Group 3 Study CaGroup 3 Study C	Group 3 Study CaGroup 3 Study C

Male	Male	Male

CM-01	CM-02	CM-03	Mean	SD

CO2 Trap 1	0-1 h	0.4472	0.4165	0.3813	0.415	0.033

CO2 Trap 1	1-3 h	*0.0174	1.1806	1.0756	0.758	0.643

CO2 Trap 1	3-6 h	3.0727	3.5843	3.5934	3.417	0.298

CO2 Trap 1	6-24 h	12.2670	11.2350	12.2830	11.928	0.600

CO2 Trap 1	24-48 h	0.8406	0.6149	1.1934	0.883	0.292

CO2 Trap 1	48-72 h	0.4159	0.2429	0.3996	0.353	0.096

CO2 Trap 1	72-96 h	0.1860	0.1479	0.2059	0.180	0.029

CO2 Trap 1	96-120 h	0.1496	0.1047	0.1822	0.146	0.039

CO2 Trap 1	120-144 h	0.1002	0.1007	0.1460	0.116	0.026

CO2 Trap 1	144-168 h	*0.0678	*0.0658	0.0841	0.073	0.010

Subtotal

17.564	17.693	19.545	18.267	1.108

a CM-04 is excluded from the table, since exhaled breath traps were saved for possible future analysis.

* Value is below 3 x background for the sample.

Table   SEQ Table \* ARABIC  15 .	  Recovery of Radioactivity in CO2 Trap 2 following exposure to 3600 ppm [2-14C]DIPE/13C6DIPE (Group 3)

Group 3 Study CaGroup 3 Study C	Group 3 Study CaGroup 3 Study C	Group 3 Study CaGroup 3 Study C	Group 3 Study CaGroup 3 Study C	Group 3 Study CaGroup 3 Study C

Male	Male	Male

CM-01	CM-02	CM-03	Mean	SD

CO2 Trap 2	0-1 h	*0.0006	*0.0021	*0.0019	0.002	0.001

CO2 Trap 2	1-3 h	1.2647	*0.0112	*0.0089	0.428	0.724

CO2 Trap 2	3-6 h	*0.0134	*0.0216	0.0273	0.021	0.007

CO2 Trap 2	6-24 h	0.9221	0.5623	1.2530	0.912	0.345

CO2 Trap 2	24-48 h	0.2068	0.2278	0.4163	0.284	0.115

CO2 Trap 2	48-72 h	0.1094	0.1368	0.1339	0.127	0.015

CO2 Trap 2	72-96 h	0.0584	0.0769	0.0691	0.068	0.009

CO2 Trap 2	96-120 h	0.0383	0.0673	0.0367	0.047	0.017

CO2 Trap 2	120-144 h	*0.0402	*0.0372	*0.0437	0.040	0.003

CO2 Trap 2	144-168 h	*0.0332	*0.0290	*0.0357	0.033	0.003

Subtotal

2.687	1.172	2.027	1.962	0.760

a CM-04 is excluded from the table, since exhaled breath traps were saved for possible future analysis.

* Value is below 3 x background for the sample.

Table   SEQ Table \* ARABIC  16 .	  Recovery of Radioactivity in Exhaled VOC Trap 1 following exposure to 3600 ppm [2-14C]DIPE/13C6DIPE (Group 3)

Group 3 Study CaGroup 3 Study C	Group 3 Study CaGroup 3 Study C	Group 3 Study CaGroup 3 Study C	Group 3 Study CaGroup 3 Study C	Group 3 Study CaGroup 3 Study C

Male	Male	Male

CM-01	CM-02	CM-03	Mean	SD

Exhaled VOC Trap 1	0-1 h	29.4100	34.3340	28.5230	30.756	3.131

Exhaled VOC Trap 1	1-3 h	18.2220	21.0790	19.8230	19.708	1.432

Exhaled VOC Trap 1	3-6 h	12.9700	12.7320	13.9460	13.216	0.643

Exhaled VOC Trap 1	6-24 h	4.1053	1.2148	5.6436	3.655	2.249

Exhaled VOC Trap 1	24-48 h	0.0488	0.0364	0.0668	0.051	0.015

Exhaled VOC Trap 1	48-72 h	0.0191	0.0258	0.0183	0.021	0.004

Exhaled VOC Trap 1	72-96 h	0.0090	0.0190	0.0098	0.013	0.006

Exhaled VOC Trap 1	96-120 h	*0.0062	0.0103	*0.0063	0.008	0.002

Exhaled VOC Trap 1	120-144 h	*0.0046	*0.0081	*0.0046	0.006	0.002

Exhaled VOC Trap 1	144-168 h	*0.0038	*0.0059	*0.0039	0.005	0.001

Subtotal

64.799	69.466	68.045	67.436	2.392

a CM-04 is excluded from the table, since exhaled breath traps were saved for possible future analysis.

* Value is below 3 x background for the sample.

Table   SEQ Table \* ARABIC  17 .	  Recovery of Radioactivity in Exhaled VOC Trap 2 following Exposure to 3600 ppm [2-14C]DIPE/13C6DIPE (Group 3)

Group 3 Study CaGroup 3 Study C	Group 3 Study CaGroup 3 Study C	Group 3 Study CaGroup 3 Study C	Group 3 Study CaGroup 3 Study C	Group 3 Study CaGroup 3 Study C

Male	Male	Male

CM-01	CM-02	CM-03	Mean	SD

Exhaled VOC Trap 2	0-1 h	*0.0017	*0.0030	*0.0028	0.003	0.001

Exhaled VOC Trap 2	1-3 h	*0.0033	*0.0040	*0.0039	0.004	0.000

Exhaled VOC Trap 2	3-6 h	*0.0013	*0.0058	*0.0048	0.004	0.002

Exhaled VOC Trap 2	6-24 h	5.4265	3.8723	0.0000	3.100	2.795

Exhaled VOC Trap 2	24-48 h	*0.0032	*0.0050	0.5099	0.173	0.292

Exhaled VOC Trap 2	48-72 h	*0.0007	*0.0007	*0.0003	0.001	0.000

Exhaled VOC Trap 2	72-96 h	*0.0006	*0.0005	0.0000	0.000	0.000

Exhaled VOC Trap 2	96-120 h	*0.0005	0.0003	0.0000	0.000	0.000

Exhaled VOC Trap 2	120-144 h	0.0001	*0.0006	0.0000	0.000	0.000

Exhaled VOC Trap 2	144-168 h	0.0002	*0.0002	0.0004	0.000	0.000

Subtotal

5.438	3.893	0.522	3.284	2.514

a CM-04 is excluded from the table, since exhaled breath traps were saved for possible future analysis.

* Value is below 3 x background for the sample.

Table   SEQ Table \* ARABIC  18 .	  Cumulative Recovery of Radioactivity in Excreta and Carcass following Exposure to 3600 ppm [2-14C]DIPE/13C6DIPE (Group 3)

Timepoint	CM-01	CM-02	CM-03	Mean	SD

0 ha	0.2368	0.1873	0.4667	0.297	0.149

1 h	30.0960	34.9430	29.3760	31.472	3.028

3 h	49.6040	57.2180	50.2870	52.370	4.213

6 h	65.6610	73.5620	67.8580	69.027	4.078

8 h	68.7440	76.5560	70.8130	72.037	4.047

24 h	94.0300	94.7010	92.5130	93.748	1.121

48 h	95.5530	95.9430	95.0980	95.531	0.423

72 h	96.3570	96.5550	95.7950	96.236	0.394

96 h	96.7090	96.8930	96.2450	96.616	0.334

120 h	96.9650	97.1510	96.5390	96.885	0.314

144 h	97.1670	97.3460	96.7640	97.092	0.298

168 h	100.0000	100.0000	100.0000	100.000	0.000

a Values at 0 h are from the samples collected at the end of the inhalation exposure, incuding the nose only tube wash and any urine and feces samples collected from the inhalation exposure.

Table   SEQ Table \* ARABIC  19 .	  Recovery of Radioactivity in Tissues from Rat CM-02 following Exposure to 3600 ppm [2-14C]DIPE/13C6DIPE (Group 3)

Timepoint	Tissue	% of Dose in Tissue

CM-02               	Blood               	0.001

CM-02               	Spleen              	0.005

CM-02               	Liver               	0.119

CM-02               	Kidney              	0.024

CM-02               	Brain               	0.014

CM-02               	Heart               	0.007

CM-02               	Lung                	0.012

CM-02               	Stomach             	0.011

CM-02               	Small Intestine     	0.026

CM-02               	Femur               	0.006

CM-02               	Testes              	0.026

CM-02               	Cecum+Lg Int+Rect   	0.019

CM-02               	Adip (Abdominal)    	0.006

CM-02               	Cage Rinse          	0.039

CM-02               	Skin (Neck)         	0.005

CM-02               	Adipose (subcut)    	0.006

CM-02               	Stomach Contents    	0.001

CM-02               	Muscle Gastrocnemius	0.004

CM-02               	Small Intestine Contents	0.019

Table   SEQ Table \* ARABIC  20 .	  Normalized Recovery of Radioactivity in Exposure B and C (Groups 2 and 3).

Exposure	Mean Exposure Concentration (ppm)	Exposure Duration (hr)	Actual ppm.hr	Recovered Dose (mg/kg)	Normalized Recovered Dose (mg/kg/ppm.hr) Normalized % Recovered

B	3529	6	21174	372.99	0.017615

	C	3334	5.66	18870	317.89	0.016846	95.6%

Figure   SEQ Figure \* ARABIC  1 .  Headspace GC-MS analysis of DIPE, acetone and isopropanol in blood.

Selected ion monitoring with total ion chromatogram (top) and (in order) acetone, 1-propanol (internal standard), DIPE, MTBE (internal standard) and isopropanol.

Figure   SEQ Figure \* ARABIC  2 .  Concentration of DIPE in male and female rat blood during and following exposure to 3600 ppm DIPE for 6 hours.

Figure   SEQ Figure \* ARABIC  3 .  Concentration of Isopropanol in male and female rat blood during and  following exposure to 3600 ppm DIPE for 6 hours.

Figure   SEQ Figure \* ARABIC  4 .  Concentration of Acetone  in male and female blood during and following exposure to 3600 ppm DIPE for 6 hours.

Figure   SEQ Figure \* ARABIC  5 .  Noncompartmental analysis of DIPE in male (top) and female (bottom) rat blood during and following exposure to 3600 ppm DIPE for 6 hours.

Figure   SEQ Figure \* ARABIC  6 .  Noncompartmental analysis of isopropanol in male (top) and female (bottom) rat blood during and following exposure to 3600 ppm DIPE for 6 hours.

Figure   SEQ Figure \* ARABIC  7 .  Noncompartmental analysis of acetone in male (top) and female (bottom) rat blood during and following exposure to 3600 ppm DIPE for 6 hours.

Figure   SEQ Figure \* ARABIC  8 .  Cumulative Excretion of Radioactivity in Urine and Feces, CO2 and Exhaled VOC Traps.

Appendix A

Approved Study Protocol, Amendments, and Deviations

RTI Protocol No. RTI-934 (RTI Project No.<PHONE_NUMBER>01)

Deviations from the Approved Protocol

The deviations listed below did not affect the results of the study.

Deviation

For Study C, animals received 3600 ppm nose-only inhalation  exposures for ca. five hours and forty minutes instead of six hours as stated in the protocol.  Actual exposure animal exposure lengths for individual animals are detailed in the study record.

Reason for the Deviation

Insufficient volume of 14C DIPE/13C DIPE formulation was available for a six-hour exposure.

Deviation

The protocol states (section 8.1) that:  “Cannula patency will be maintained with daily flushings of heparinized saline (sterile saline containing  20 IU/mL of sodium heparin).  Jugular cannulas will not be flushed with heparinized saline prior to use on exposure day.

Reason for the Deviation

The vendor (Charles River Laboratories) specified that the cannulas retain patency best if the heparinized solution used to lock the cannula is not disturbed until use (up through  one week prior to use).

Deviation

Aliquots of the Study B and Study C inhalation formulatins were transferred to a single vial  (for each formulation) already containing 20 mL of dimethylformamide instead of an empty vial.  Weights of )1 empty vial, 2) vial+DMF, and 3) weight of vial+DMF+formulation aliquot were recorded in the process.  Aliquots of the DMF dilution wiere then transferred to vials already containing scintillation cocktail for LSS analysis instead of adding cocktail after the transfer.

 Reason for the Deviation

At the time, concern about evaporation of the DIPE formulation during transfer to empty vials, thereby affecting quantiation results.  Addition of DIPE formulation or the dilution to solvent was expected to minimize evaporation.

.Appendix B

Test Chemical Analysis Report

Appendix C

Test Chemical Analysis Report:  2-14C Diisopropyl ether

Test Chemical Analysis Report:  13C6-Diisopropyl ether

Analysis of Reference Substances: Acetone and Isopropanol

Appendix D

Method Validation for Analysis of DIPE, Isopropanol and Acetone

Appendix E

Pre-Study Inhalation Report

Appendix F

Inhalation Reports

Appendix G

Example CalculationsFormulae for Calculations

Formulae used in the Debra system for calculations are shown below:

Aliquot dpm/g is calculated by:

dpm/g = aliquot corrected count (dpm) / aliquot weight (g)

The mean of these aliquot figures for the sample is calculated by:

Mean dpm/g = Sum of all aliquot dpm/g / number of aliquots

The weight of the sample used for calculations is calculated from the pot weight (before sample collection) and sample weight (weight of pot + sample):

Weight of the sample = sample weight – pot weight

Depending on the nature of the sample processing, Effective weight may be one of a number of values:

If processing type does not use homogenate (e.g. for urine, exhaled VOC trap, CO2 trap, Cage Wash) then:

Effective weight = weight of the sample

If processing type uses homogenate (e.g. for feces and carcass) then:

Effective weight = Homogenate weight

To calculate the total dpm in the sample, Debra uses the previously calculated figures;

Total Dpm = Mean dpm/g * Effective weight

For inhalation exposure, where the dpm administered is not precisely known, the Dose Dpm is calculated as the sum of all dpm recovered for each animal.

Recovery is calculated by:

Recovery = 100* (Total Dpm/ Dose Dpm)

Concentration is calculated by:

Concentration = (Total dpm/ effective weight) / (Specific activity)

Results are expressed in terms of the arithmetic mean ( standard deviation. Mean values were calculated using:

Standard deviation was calculated using:

Example Calculations

Examples of the calculations for urine, feces, exhaled VOC trap, CO2 trap, and cage rinse made in the Debra™ system are shown below:

Subject		= CM-02

Tissue		= Urine

Timepoint	= 24 h

Aliquot 1 dpm/g	= 9129.865/0.0532

		=<PHONE_NUMBER>59398 dpm/g

Aliquot 2 dpm/g	= 9167.455/0.053

		=<PHONE_NUMBER>56604 dpm/g

Sum of dpm/g	=<PHONE_NUMBER>16002 dpm/g

Number of aliquots	= 2

Mean dpm/g	= Sum of dpm/g / Number of aliquots

		=<PHONE_NUMBER>16002 / 2

		=<PHONE_NUMBER>08001 dpm/g

		= 0.393762316049851 %

Recovery Effective Wt

		= Sample weight

		= 2.9801g

Concentration Effective Weight

		= Sample weight

		= 2.9801g

Total dpm		= Dpm/g x Recovery effective weight

		=<PHONE_NUMBER>08001 x 2.9801

		=<PHONE_NUMBER>38484 dpm

Dose dpm (using Exposure Dose)

		= sum of all dpm recovered

		= 57600304.4347919 dpm

Recovery		= 100 x (Total dpm / Dose dpm)

		= 100 x<PHONE_NUMBER>38484 / 57600304.4347919)

		= 100 x (0.00891399212168659)

		= 0.891399212168659%

Concentration	= (Total dpm/Concentration effective weight) / (Specific activity)

		=<PHONE_NUMBER>38484 / 2.9801) / (0.4241438147 µCi/mg)

		=<PHONE_NUMBER>08001 dpm/g) /<PHONE_NUMBER>34 dpm/mg)

		=<PHONE_NUMBER>19561 µg/g

Subject		= CM-02

Tissue		= Feces

Timepoint	= 24 h

Aliquot 1 dpm/g	= 1953.942/0.082

		= 23828.5609756098 dpm/g

Aliquot 2 dpm/g	= 2254.492/0.0927

		= 24320.3020496224 dpm/g

Sum of dpm/g	= 48148.8630252322 dpm/g

Number of aliquots	= 2

Mean dpm/g	= Sum of dpm/g / Number of aliquots

		= 48148.8630252322 / 2

		= 24074.4315126161 dpm/g

Recovery Effective Weight

		= Homogenate weight

		= 8.8412g

Concentration Effective Weight

		= Sample weight

		= 4.24769999999999g

Total dpm		= Dpm/g x Recovery effective weight

		= 24074.4315126161 x 8.8412

		=<PHONE_NUMBER>89341 dpm

Dose dpm (using Exposure Dose)

		= sum of all dpm recovered

		= 57600304.4347919 dpm

Recovery		= 100 x (Total dpm / Dose dpm)

		= 100 x<PHONE_NUMBER>89341 / 57600304.4347919)

		= 100 x (0.00369523852309324)

		= 0.369523852309324%

Concentration	= (Total dpm/Concentration effective weight) / (Specific activity)

		=<PHONE_NUMBER>89341 / 4.24769999999999) / (0.4241438147 µCi/mg)

		= (50108.7326998945 dpm/g) /<PHONE_NUMBER>34 dpm/mg)

		= 53.2166223669527 µg/g

Subject		= CM-02

Tissue		= Exhaled VOC Trap 1

Timepoint	= 1 h

Aliquot 1 dpm/g	=<PHONE_NUMBER>5/0.0482

		= 2511577.04875519 dpm/g

Aliquot 2 dpm/g	=<PHONE_NUMBER>5/0.0469

		= 2592409.46162047 dpm/g

Sum of dpm/g	= 5103986.51037566 dpm/g

Number of aliquots	= 2

Mean dpm/g	= Sum of dpm/g / Number of aliquots

		= 5103986.51037566 / 2

		= 2551993.25518783 dpm/g

Recovery Effective Wt

		= Sample weight

		= 7.7495g

Concentration Effective Weight

		= Sample weight

		= 7.7495g

Total dpm		= Dpm/g x Recovery effective weight

		= 2551993.25518783 x 7.7495

		= 19776671.7310781 dpm

Dose dpm (using Exposure Dose)

		= sum of all dpm recovered

		= 57600304.4347919 dpm

Recovery		= 100 x (Total dpm / Dose dpm)

		= 100 x (19776671.7310781 / 57600304.4347919)

		= 100 x (0.343343180650492)

		= 34.3343180650492%

Concentration	= (Total dpm/Concentration effective weight) / (Specific activity)

		= (19776671.7310781 / 7.7495) / (0.4241438147 µCi/mg)

		= (2551993.25518783 dpm/g) /<PHONE_NUMBER>34 dpm/mg)

		= 2710.27531583586 µg/g

Subject		= CM-02

Tissue		= CO2 Trap 1

Timepoint	= 1 h

Aliquot 1 dpm/g	=<PHONE_NUMBER>33333/1.064

		=<PHONE_NUMBER>0802 dpm/g

Aliquot 2 dpm/g	=<PHONE_NUMBER>33333/1.031

		=<PHONE_NUMBER>0915 dpm/g

Sum of dpm/g	=<PHONE_NUMBER>1717 dpm/g

Number of aliquots	= 2

Mean dpm/g	= Sum of dpm/g / Number of aliquots

		=<PHONE_NUMBER>1717 / 2

		=<PHONE_NUMBER>58585 dpm/g

Recovery Effective Wt

		= Sample weight

		= 523.6g

Concentration Effective Weight

		= Sample weight

		= 523.6g

Total dpm		= Dpm/g x Recovery effective weight

		=<PHONE_NUMBER>58585 x 523.6

		=<PHONE_NUMBER>90675 dpm

Dose dpm (using Exposure Dose)

		= sum of all dpm recovered

		= 57600304.4347919 dpm

Recovery		= 100 x (Total dpm / Dose dpm)

		= 100 x<PHONE_NUMBER>90675 / 57600304.4347919)

		= 100 x (0.00416451694734071)

		= 0.416451694734071%

Concentration	= (Total dpm/Concentration effective weight) / (Specific activity)

		=<PHONE_NUMBER>90675 / 523.6) / (0.4241438147 µCi/mg)

		=<PHONE_NUMBER>58585 dpm/g) /<PHONE_NUMBER>34 dpm/mg)

		= 0.486545726318592 µg/g

Subject		= CM-02

Tissue		= Exhaled VOC Trap 1

Timepoint	= 168 h

Aliquot 1 dpm/g	= 42.3866666666667/0.049

		=<PHONE_NUMBER>05443 dpm/g

Aliquot 2 dpm/g	= 40.9566666666667/0.0464

		=<PHONE_NUMBER>09196 dpm/g

Sum of dpm/g	= 1747.72079521464 dpm/g

Number of aliquots	= 2

Mean dpm/g	= Sum of dpm/g / Number of aliquots

		= 1747.72079521464 / 2

		=<PHONE_NUMBER>07319 dpm/g

Sum of (dpm/g)^2	= (Aliquot 1 dpm/g)^2 + .. + (Aliquot n dpm/g)^2

		= 1527419.79912194

SD of dpm/g	= Sqrt((n x Sum of (dpm/g)^2-(Sum of dpm/g)^2) / n(n-1))

		= Sqrt((Sum of (dpm/g)^2-((Sum of dpm/g)^2)/n) / (n-1))

		= Sqrt(1527419.79912194 - (3054527.97802569 / 2) / (2-1))

		= 12.4823919621806

Dpm/g %Variance (2 aliquot rule)

		= 100 x ((Mean dpm/g - aliquot 1 dpm/g) / Mean dpm/g)

		= 100 x ((873.860397607319 -<PHONE_NUMBER>05443) /<PHONE_NUMBER>07319)

		= 100 x 0.0101004508569603

		= 1.01004508569603 %

Recovery Effective Wt

		= Sample weight

		= 3.894g

Concentration Effective Weight

		= Sample weight

		= 3.894g

Total dpm		= Dpm/g x Recovery effective weight

		=<PHONE_NUMBER>07319 x 3.894

		= 3402.8123882829 dpm

Dose dpm (using Exposure Dose)

		= sum of all dpm recovered

		= 57600304.4347919 dpm

Recovery		= 100 x (Total dpm / Dose dpm)

		= 100 x (3402.8123882829 / 57600304.4347919)

		= 100 x (5.90762917257695E-5)

		= 0.00590762917257695%

Recovery SD	= Recovery x (dpm/g SD / dpm/g)

		= 0.00590762917257695 x (12.4823919621806 /<PHONE_NUMBER>07319)

		= 0.00590762917257695 / 0.0142841945880121

		= 8.43857246549062E-5

Concentration	= (Total dpm/Concentration effective weight) / (Specific activity)

		= (3402.8123882829 / 3.894) / (0.4241438147 µCi/mg)

		=<PHONE_NUMBER>07319 dpm/g) /<PHONE_NUMBER>34 dpm/mg)

		= 0.928059766736065 µg/g

Concentration SD	= Concentration x (dpm/g SD / dpm/g)

		= 0.000928059766736065 mg/g x (12.4823919621806 /<PHONE_NUMBER>07319)

		= 0.000928059766736065 / 0.0142841945880121

		= 0.0132565862973631

Subject		= CM-02

Tissue		= Cage Rinse

Timepoint	= 168 h

Aliquot 1 dpm/g	= 60.16/0.5662

		=<PHONE_NUMBER>00459 dpm/g

Aliquot 2 dpm/g	= 67.4/0.5789

		=<PHONE_NUMBER>21541 dpm/g

Sum of dpm/g	=<PHONE_NUMBER>22 dpm/g

Number of aliquots	= 2

Mean dpm/g	= Sum of dpm/g / Number of aliquots

		=<PHONE_NUMBER>22 / 2

		=<PHONE_NUMBER>11 dpm/g

Recovery Effective Wt

		= Sample weight

		= 202.8g

Concentration Effective Weight

		= Sample weight

		= 202.8g

Total dpm		= Dpm/g x Recovery effective weight

		=<PHONE_NUMBER>11 x 202.8

		= 22579.7434237908 dpm

Dose dpm (using Exposure Dose)

		= sum of all dpm recovered

		= 57600304.4347919 dpm

Recovery		= 100 x (Total dpm / Dose dpm)

		= 100 x (22579.7434237908 / 57600304.4347919)

		= 100 x (0.00039200736255401)

		= 0.039200736255401%

Recovery SD	= Recovery x (dpm/g SD / dpm/g)

		= 0.039200736255401 x (7.1951650668709 /<PHONE_NUMBER>11)

		= 0.039200736255401 / 0.0646233860223573

		= 0.0025332843113934

Concentration	= (Total dpm/Concentration effective weight) / (Specific activity)

		= (22579.7434237908 / 202.8) / (0.4241438147 µCi/mg)

		=<PHONE_NUMBER>11 dpm/g) /<PHONE_NUMBER>34 dpm/mg)

		= 0.118245586439891 µg/g

Appendix H

Raw Data Tables for Radioactivity

Sample Naming and Abbreviations

In the tables of raw data values in this appendix, the table headings are derived from the conventions of sample naming used in the Debra( system.  These are explained below.  Example calculations and formulae are provided in Appendix G.

Column Heading	Source	Explanation

Subject

Animal number

Sample

The type of sample, e.g. urine, feces

Time

The time of sample collection

Pot wt	Balance	The weight of the empty sample container

Samp wt	Balance	Sample weight,  the weight of sample + empty sample container

Corr Samp	Calculated	The corrected sample weight, calculated from Samp wt – Pot wt

Homog wt	Balance	For samples requiring homogenization, the weight of pot + sample + medium for homogenization.

Corr Homog	Calculated	Corrected Homogenate Weight,

calculated from Homog Wt – Samp Wt.

Alq wt	Balance	Aliquot weight, weight of aliquot removed for scintillation counting

Orig DPM	Scintillation Counter	Original DPM, the DPM measured in each aliquot.

Bkg DPM	Scintillation Counter	The background DPM determined for the batch of samples

Calc DPM	Calculated	The calculated DPM for the aliquot, = Orig DPM – Calc DPM

LOD	Calculated	Limit of Detection.  If Orig DMP value is less than Bkg DMP, then the sample is flagged with an *

LOQ	Calculated	Limit of Quantitation.  If Orig DMP value is less than 3 x Bkg DMP, then the sample is flagged with an *

DPM/g	Calculated	Calculated value of DPM/g sample.

Sample DPM	Calculated	Total DPM in the sample.

Reco (%)	Calculated	Percentage of the total recovered radioactivity present in the sample.

Sample DPM Sum	Calculated	The sum of radioactivity in all samples for a subject.

Table 1.  Preparation of 14C DIPE Formulation (Feedstock) for Inhalation Exposure

14C DIPE for Study B

Ci/mg	(J)=(I)*1000

Nominal Specific Activity	38.76798779	Ci/mmol (K)=(((H)*1000)/(G))*(B)

	Formulation Dilution

Weight of Feedstock 	0.0679	g	(L)

Weight of DMF	20.4687	g	(M)

Total weight of solution	20.5366	g	(N)=(L)+(M)

	Dose Formulation Dilution Aliquots

	Aliq #	Aliquot Wt (g)	DPM	DPM/g

	1	0.0876	301093	3437135

2	0.0862	291199	3378179

3	0.0955	295976	3099225

	Mean dilution DPM/g

	3304846

Ci/g	(R)=(Q)/2220000

Specific Activity (Ci/mg DIPE)	0.450253444	Ci/mg  S)=(R)*(F)/(G)

Specific Activity (Ci/mmol DIPE)	46.00689695	Ci/mmol  (T)=(R)*(B)/1000

Table 1 (contd).  Preparation of 14C DIPE Formulation (Feedstock) for Inhalation Exposure

13C/14C DIPE For Study C

Ci/g	(R)=(Q)/2220000

Specific Activity (Ci/mg DIPE)	0.424143815	Ci/mg  S)=(R)*(F)/(G)

Specific Activity (Ci/mmol DIPE)	45.88387787	Ci/mmol  (T)=(R)*(B)/1000

Table 2.  Recovery of Radioactivity in Samples from Group B.

Subject	Sample	Time	Pot wt	Samp wt	Corr Samp	Homog wt	Corr Homog	Alq wt Orig DPM	Bkg DPM	Calc DPM	LOD	LOQ	Mean DPM/g	Sample DPM	Recovery (%)

BM-01

	Carcass Digest      	  0 h

	109.00

	299.11

	190.11

	740.66

	631.66

	0.1074

0.1048	12678.60

12836.32	26.41

26.41	12652.190

12809.910

	120018.171

	75810677.8347

	98.200

BM-01

	Exhaled VOC         	  0 h

	17.0963

	24.8570

	7.7607

	N.A.

	N.A.

	0.0447

0.0460	2854.75

2879.17	25.67

25.67	2829.084

2853.504

	62661.577

<PHONE_NUMBER>

	0.630

BM-01

	Exposure Urine      	  0 h

	16.3491

	Missing

	Missing

	N.A.

	N.A.

	N.C.

N.C.	N.C.

N.C.	N.C.

N.C.	N.A.

N.A.

	N.A.

	N.C.

	N.A.

BM-01

	Exposure Feces      	  0 h

	16.5629

	22.1174

	5.5545

	23.7509

	7.1880

	0.0636

0.0668	6646.67

6672.22	9.66

9.66	6637.007

6662.557

	102047.161

<PHONE_NUMBER>

	0.950

BM-01

	Nose Only Tube Rinse	  0 h

	107.09

	301.24

	194.15

	N.A.

	N.A.

	0.4110

0.4885

0.4619	357.85

429.17

416.26	4.86

4.86

4.86	352.990

424.310

411.400

	872.708

<PHONE_NUMBER>

	0.219

BM-02

	Carcass Digest      	  0 h               	108.94

	303.89

	194.95

	751.53

	642.59

	0.1053

0.1150	11916.17

13281.38	26.41

26.41	11889.760

13254.970

	114086.905

	73311103.9869

	97.751

BM-02

	Exhaled VOC         	  0 h

	17.3547

	25.0557

	7.7010

	N.A.

	N.A.

	0.0456

0.0468	5250.95

5340.78	25.67

25.67	5225.284

5315.114

	114080.181

<PHONE_NUMBER>

	1.171

BM-02

	Exposure Urine      	  0 h

	16.5909

	Missing

	Missing

	N.A.

	N.A.

	N.C.

N.C.	N.C.

N.C.	N.C.

N.C.	N.A.

N.A.

	N.A.

	N.C.

	N.A.

BM-02

	Exposure Feces      	  0 h

	16.3308

	20.9757

	4.6449

	22.5426

	6.2118

	0.1079

0.0662	12210.99

7020.68	9.66

9.66	12201.327

7011.017

	109493.273

<PHONE_NUMBER>

	0.907

BM-02

	Nose Only Tube Rinse	  0 h

	108.03

	271.25

	163.22

	N.A.

	N.A.

	0.5193

0.4742

0.4911	404.39

367.04

406.95	4.86

4.86

4.86	399.530

362.180

402.090

783.962

<PHONE_NUMBER>

	0.171

BM-03

	Carcass Digest      	  0 h

	108.55

	304.29

	195.74

	753.38

	644.83

	0.1089

0.1076	12673.66

12160.60	26.41

26.41	12647.250

12134.190

	114453.823

	73803258.7380

	99.529

BM-03

	Exhaled VOC         	  0 h               	17.2584	25.0178	7.7594	N.A. N.A.	0.0474

0.0476	21.73

23.28	28.57

28.57	0.000

0.000	*

*

	0.000

	0.0000

	0.000

BM-03

	Exposure Urine      	  0 h

	16.7684

	Missing

	Missing

	N.A.

	N.A.

	N.C.

N.C.	N.C.

N.C.	N.C.

N.C.	N.A.

N.A.

	N.A.

	N.C.

	N.A.

BM-03

	Exposure Feces      	  0 h

	16.1935

	18.1833

	1.9898

	19.7737

	3.5802

	0.0723

0.0856	3400.14

4134.72	9.66

9.66	3390.477

4125.057

	47542.237

<PHONE_NUMBER>

	0.230

BM-03

	Nose Only Tube Rinse	  0 h

	107.42

	261.91

	154.49

	N.A.

	N.A.

	0.6756

0.4754

0.5117	800.72

554.35

586.39	4.86

4.86

4.86	795.860

549.490

581.530

	1156.773

<PHONE_NUMBER>

	0.241

BM-04

	Carcass Digest      	  0 h

	109.11

	296.36

	187.25

	719.52

	610.41

	0.0922

0.0927	11057.51

10924.22	26.41

26.41	11031.100

10897.810

	118601.573

	72395586.0081

	98.726

BM-04

	Exhaled VOC         	  0 h

	17.2089

	25.0106

	7.8017

	N.A.

	N.A.

	0.0458

0.0448	2202.97

2249.47	25.67

25.67	2177.304

2223.804

	48588.930

<PHONE_NUMBER>

	0.517

BM-04

	Exposure Urine      	  0 h

	16.6977

	Missing

	Missing

	N.A.

	N.A.

	N.C.

N.C.	N.C.

N.C.	N.C.

N.C.	N.A.

N.A.

	N.A.

	N.C.

	N.A.

BM-04

	Exposure Feces      	  0 h

	16.7245

	20.0300

	3.3055

	23.1542

	6.4297

	0.0788

0.0934	3576.95

3938.10	9.66

9.66	3567.287

3928.437

	43665.243

<PHONE_NUMBER>

	0.383

BM-04

	Exp. Urine Sample 2 	  0 h

	107.82

	121.65

	13.83

	625.14

	517.32

	0.9189

0.9323

0.6153	277.92

278.43

188.89	4.88

4.88

4.88	273.040

273.550

184.010

	296.536

<PHONE_NUMBER>

	0.209

BM-04

	Nose Only Tube Rinse

106.88

	184.10

	77.22

	N.A.

	N.A.

	0.4604

0.5673

0.6552	738.94

911.27

987.73	4.86

4.86

4.86	734.080

906.410

982.870

	1564.103

<PHONE_NUMBER>

	0.165

* in LOD column indicates that sample DPM were less than background DPM, and in LOQ column indicates that sample DPM was less than 3 x background.

Table 3.  Total Recovery of Radioactivity in Samples from Group B.

Subject	Sample	Time	Sample DPM	Sample DPM Sum	Recovery (%)

1-01	Feces	0 h<PHONE_NUMBER> dpm	59110333.860 dpm	0.215

1-01	Carcass Digest	0 h	58796748.8875 dpm

99.469

1-01	Exhaled VOC	0 h	1110.6238 dpm

0.002

1-01	Nose Only Tube Rinse	0 h<PHONE_NUMBER> dpm

0.281

1-01	Transfer Bag Rinse	0 h	18936.7872 dpm

0.032

1-02	Feces	0 h	24901.9839 dpm	60334744.156 dpm	0.041

1-02	Carcass Digest	0 h	60107213.0993 dpm

99.623

1-02	Exhaled VOC	0 h	785.6933 dpm

0.001

1-02	Nose Only Tube Rinse	0 h<PHONE_NUMBER> dpm

0.329

1-02	Transfer Bag Rinse	0 h	3112.3069 dpm

0.005

1-03	Feces	0 h<PHONE_NUMBER> dpm	61168290.196 dpm	0.244

1-03	Carcass Digest	0 h	60412728.2023 dpm

98.765

1-03	Exhaled VOC	0 h	2464.3752 dpm

0.004

1-03	Nose Only Tube Rinse	0 h<PHONE_NUMBER> dpm

0.426

1-03	Transfer Bag Rinse	0 h<PHONE_NUMBER> dpm

0.561

1-04	Feces	0 h<PHONE_NUMBER> dpm	49730780.503 dpm	1.670

1-04	Carcass Digest	0 h	48198191.8677 dpm

96.918

1-04	Exhaled VOC	0 h	8048.3908 dpm

0.016

1-04	Nose Only Tube Rinse	0 h<PHONE_NUMBER> dpm

1.362

1-04	Transfer Bag Rinse	0 h	16572.4584 dpm

0.033

Subject 	Sample 	Time 	Sample dpm 	Sample dpm Sum	Reco (%)

BM-01               	Carcass Digest	  0 h               	75810677.83 dpm 77199926.730 dpm 	98.200

BM-01               	Exhaled VOC	  0 h               	486297.70 dpm

0.630

BM-01               	Exposure Feces	  0 h               	733515.00 dpm

0.950

BM-01               	Nose Only Tube Rinse	  0 h               	169436.20 dpm

0.219

BM-02               	Carcass Digest	  0 h               	73311103.99 dpm 74997744.120 dpm 	97.751

BM-02               	Exhaled VOC	  0 h               	878531.48 dpm

1.171

BM-02               	Exposure Feces	  0 h               	680150.32 dpm

0.907

BM-02               	Nose Only Tube Rinse	  0 h               	127958.33 dpm

0.171

BM-03               	Carcass Digest	  0 h               	73803258.74 dpm 74152179.330 dpm 	99.529

BM-03               	Exhaled VOC	  0 h               	0.00 dpm

0.000

BM-03               	Exposure Feces	  0 h               	170210.72 dpm

0.230

BM-03               	Nose Only Tube Rinse	  0 h               	178709.87 dpm

0.241

BM-04               	Carcass Digest	  0 h               	72395586.01 dpm 73329600.910 dpm 	98.726

BM-04               	Exhaled VOC	  0 h               	379076.25 dpm

0.517

BM-04               	Exposure Feces	  0 h               	280754.41 dpm

0.383

BM-04               	Exp. Urine Sample 2	  0 h               	153404.24 dpm

0.209

BM-04               	Nose Only Tube Rinse	  0 h               	120780.00 dpm

0.165

Table 4.  Recovery of Radioactivity in Urine from Group C.

Subject	Sample	Time	Pot wt	Samp wt	Corr Samp	Alq wt	Orig DPM	Bkg DPM Calc DPM	LOD	LOQ	DPM/g	Sample DPM	Reco (%)

CM-01

	Urine

	  8 h

	16.3833 g

	20.0443 g

	3.6610 g

	0.0538 g

0.0535 g	25207.96

24753.04	25.83

25.83	25182.130

24727.210

	468069.331

462190.841	1702841.2448

	3.083

CM-01

	Urine

	 24 h

	49.6715 g

	54.2445 g

	4.5730 g

	0.0546 g

0.0539 g	13386.21

13071.36	31.37

31.37	13354.845

13039.995

	244594.231

241929.406	1112436.2962

	2.014

CM-01

	Urine

	 48 h

	49.8979 g

	54.5841 g

	4.6862 g

	0.1980 g

0.2073 g	6452.76

6718.38	4.60

4.60	6448.165

6713.785

	32566.490

32386.807<PHONE_NUMBER>

	0.276

CM-01

	Urine

	 72 h

	50.0245 g

	57.9905 g

	7.9660 g

	0.2080 g

0.2110 g	1112.64

1144.74	3.90

3.90	1108.740

1140.840

	5330.481

5406.825	42766.6875

	0.077

CM-01

	Urine

	 96 h

	49.4195 g

	58.9340 g

	9.5145 g

	0.2084 g

0.2055 g	448.37

457.20	3.90

3.90	444.470

453.300

	2132.774

2205.839	20639.8664

	0.037

CM-01

	Urine

	120 h

	49.3183 g

	60.9000 g

	11.5817 g

	0.2033 g

0.2057 g	246.10

235.53	3.90

3.90	242.200

231.630

	1191.343

1126.057	13419.7170

	0.024

CM-01

	Urine

	144 h

	49.4390 g

	58.4684 g

	9.0294 g

	0.2040 g

0.2054 g	163.49

181.32	0.00

0.00	163.490

181.320

	801.422

882.765	7603.5986

	0.014

CM-01

	Urine

	168 h

	48.4712 g

	58.6314 g

	10.1602 g

	0.2080 g

0.2077 g	138.30

139.64	3.10

3.10	135.205

136.545

	650.024

657.415	6641.9187

	0.012

CM-02

	Urine

	  8 h

	16.4290 g

	20.4321 g

	4.0031 g

	0.0542 g

0.0527 g	23333.85

22766.29	25.83

25.83	23308.020

22740.460

	430037.269

431507.780	1724425.4933

	2.994

CM-02

	Urine

	 24 h

	49.6840 g

	52.6641 g

	2.9801 g

	0.0532 g

0.0530 g	9161.23

9198.82	31.37

31.37	9129.865

9167.455

	171614.004

172970.849<PHONE_NUMBER>  	0.891

CM-02

	Urine

	 48 h

	49.5056 g

	52.9815 g

	3.4759 g

	0.1035 g

0.1029 g	2965.31

2965.80	4.60

4.60	2960.715

2961.205

	28605.942

28777.502	99729.5573

	0.173

CM-02

	Urine

	 72 h

	49.4267 g

	61.2787 g

	11.8520 g

	0.2068 g

0.2109 g	794.63

830.46	3.90

3.90	790.730

826.560

	3823.646

3919.203	45884.1258

	0.080

CM-02

	Urine

	 96 h

	50.2654 g

	69.1980 g

	18.9326 g

	0.2001 g

0.2011 g	277.13

278.05	3.90

3.90	273.230

274.150

	1365.467

1363.252	25830.8763

	0.045

CM-02

	Urine

	120 h

	49.7676 g

	58.2694 g

	8.5018 g

	0.2089 g

0.2121 g	336.87

329.49	3.90

3.90	332.970

325.590

	1593.921

1535.078	13301.0590

	0.023

CM-02

	Urine

	144 h

	47.7846 g

	56.7403 g

	8.9557 g

	0.2044 g

0.2075 g	181.95

192.33	0.00

0.00	181.950

192.330

	890.166

926.892	8136.5127

	0.014

CM-02

	Urine

	168 h

	50.0855 g

	60.5776 g

	10.4921 g

	0.2077 g

0.2101 g	161.69

174.21	3.10

3.10	158.595

171.115

	763.577

814.446	8278.3864

	0.014

* in LOD column indicates that sample DPM were less than background DPM, and in LOQ column indicates that sample DPM was less than 3 x background.

Table 4 (contd).  Recovery of Radioactivity in Urine from Group 2.

Subject	Sample	Time	Pot wt	Samp wt	Corr Samp	Alq wt	Orig DPM	Bkg DPM Calc DPM	LOD	LOQ	DPM/g	Sample DPM	Reco (%)

CM-03

	Urine

	  8 h

	16.5370 g

	21.0053 g

	4.4683 g

	0.0512 g

0.0517 g	20958.37

21257.53	25.83

25.83	20932.540

21231.700

	408838.672

410671.180	1830907.9353

	2.954

CM-03

	Urine

	 24 h

	49.3539 g

	55.0547 g

	5.7008 g

	0.0518 g

0.0540 g	9358.26

9916.82	31.37

31.37	9326.895

9885.455

	180055.888

183063.981	1035036.8761

	1.670

CM-03

	Urine

	 48 h

	50.0055 g

	59.6864 g

	9.6809 g

	0.1978 g

0.2022 g	1933.32

1965.51	4.60

4.60	1928.725

1960.915

	9750.885

9697.898	94140.8610

	0.152

CM-03

	Urine

	 72 h

	49.5513 g

	62.8301 g

	13.2788 g

	0.2049 g

0.2116 g	578.91

570.88	3.90

3.90	575.010

566.980

	2806.296

2679.490	36422.3233

	0.059

CM-03

	Urine

	 96 h

	49.3539 g

	64.2525 g

	14.8986 g

	0.2054 g

0.2046 g	296.89

301.67	3.90

3.90	292.990

297.770

	1426.436

1455.376	21467.4863

	0.035

CM-03

	Urine

	120 h

	49.2192 g

	59.1509 g

	9.9317 g

	0.2087 g

0.2067 g	247.53

264.03	3.90

3.90	243.630

260.130

	1167.369

1258.491	12046.4569

	0.019

CM-03

	Urine

	144 h

	50.0996 g

	62.7657 g

	12.6661 g

	0.2021 g

0.2055 g	181.35

176.72	0.00

0.00	181.350

176.720

	897.328

859.951	11128.9383

	0.018

CM-03

	Urine

	168 h

	50.2428 g

	59.7059 g

	9.4631 g

	0.2064 g

0.2097 g	143.11

144.77	3.10

3.10	140.015

141.675

	678.367

675.608	6406.4016

	0.010

CM-04

	Urine

	  8 h

	16.6114 g

	18.5065 g

	1.8951 g

	0.0529 g

0.0539 g	27836.60

27971.21	25.83

25.83	27810.770

27945.380

	525723.440

518467.161<PHONE_NUMBER>

	6.461

CM-04

	Urine

	 24 h

	50.0264 g

	57.2613 g

	7.2349 g

	0.0542 g

0.0524 g	10002.06

9761.64	31.37

31.37	9970.695

9730.275

	183961.162

185692.271	1337202.8125

	8.732

CM-04

	Urine

	 48 h

	49.1938 g

	65.1926 g

	15.9988 g

	0.2017 g

0.2051 g	2284.97

2395.29	4.60

4.60	2280.375

2390.695

	11305.776

11656.241<PHONE_NUMBER>  	1.199

CM-04

	Urine

	 72 h

	49.9395 g

	61.4514 g

	11.5119 g

	0.2071 g

0.2098 g	684.50

742.74	3.90

3.90	680.600

738.840

	3286.335

3521.640	39186.3623

	0.256

CM-04

	Urine

	 96 h

	48.7565 g

	56.3028 g

	7.5463 g

	0.2121 g

0.2096 g	393.63

372.46	3.90

3.90	389.730

368.560

	1837.482

1758.397	13567.7919

	0.089

CM-04

	Urine

	120 h

	49.9636 g

	60.1770 g

	10.2134 g

	0.2100 g

0.2111 g	251.37

243.74	3.90

3.90	247.470

239.840

	1178.429

1136.144	11819.8278

	0.077

CM-04

	Urine

	144 h

	49.5018 g

	58.9107 g

	9.4089 g

	0.2049 g

0.2084 g	194.47

210.64	0.00

0.00	194.470

210.640

	949.097

1010.749	9219.9960

	0.060

CM-04

	Urine

	168 h

	50.9518 g

	60.1408 g

	9.1890 g

	0.2091 g

0.2125 g	166.56

163.50	3.10

3.10	163.465

160.405

	781.755

754.847	7059.9188

	0.046

* in LOD column indicates that sample DPM were less than background DPM, and in LOQ column indicates that sample DPM was less than 3 x background.

Table 5.  Recovery of Radioactivity in Feces from Group C.

Subject	Sample	Time	Pot wt	Samp wt	Corr Samp	Homog wt	Corr Homog	Alq wt Orig DPM	Bkg DPM	Calc DPM	LOD	LOQ	DPM/g	Sample DPM	Reco (%)

CM-01

	Feces

	 24 h

	50.1379

	55.4465

	5.3086

	60.6817

	10.5438

	0.0775

0.0906 	2308.16

2566.50	16.42

16.42	2291.742

2550.082

	29570.865

28146.600<PHONE_NUMBER>  	0.551

CM-01

	Feces

	 48 h

	50.3414

	54.0328

	3.6914

	57.8248

	7.4834

	0.0859

0.0947 	962.37

1042.05	16.42

16.42	945.952

1025.632

	11012.247

10830.327	81728.3597  	0.148

CM-01

	Feces

	 72 h

	50.1662

	58.1751

	8.0089

	65.3328

	15.1666

	0.0899

0.0995 	669.00

609.90	16.42

16.42	652.582

593.482

	7258.977

5964.643<PHONE_NUMBER>  	0.182

CM-01

	Feces

	 96 h

	49.1753

	55.0041

	5.8288

	61.1627

	11.9874

	0.0599

0.0621 	176.87

182.11	9.08

9.08	167.788

173.028

	2801.127

2786.272	33489.1935  	0.061

CM-01

	Feces

	120 h

	50.2002

	55.1973

	4.9971

	60.9793

	10.7791

	0.0731

0.0547 	145.04

112.66	9.08

9.08	135.958

103.578

	1859.884

1893.556	20229.3497  	0.037

CM-01

	Feces

	144 h

	49.7822

	60.6093

	10.8271

	72.6359

	22.8537

	0.0538

0.0536 	65.59

64.40	9.08

9.08	56.508

55.318

	1050.325

1032.043	23794.9089  	0.043

CM-01

	Feces

	168 h

	50.0089

	56.5155

	6.5066

	65.8127

	15.8038

	0.0567

0.0546 	63.24

65.20	9.08

9.08	54.158

56.118

	955.159

1027.793	15669.0866  	0.028

CM-02

	Feces

	 24 h

	50.5312

	54.7789

	4.2477

	59.3724

	8.8412

	0.0820

0.0927 	1970.36

2270.91	16.42

16.42	1953.942

2254.492

	23828.561

24320.302<PHONE_NUMBER>  	0.370

CM-02

	Feces

	 48 h

	49.8609

	53.3509

	3.4900

	57.3311

	7.4702

	0.0631

0.0781 	901.67

1143.17	16.42

16.42	885.252

1126.752

	14029.350

14427.042<PHONE_NUMBER>  	0.185

CM-02

	Feces

	 72 h

	49.6047

	61.2648

	11.6601

	75.0969

	25.4922

	0.0862

0.0725 	255.68

228.62	16.42

16.42	239.262

212.202

	2775.661

2926.924	72685.7237  	0.126

CM-02

	Feces

	 96 h

	50.2877

	56.0242

	5.7365

	66.1906

	15.9029

	0.0932

0.0802 	169.07

155.96	9.08

9.08	159.988

146.878

	1716.604

1831.390	28211.6997  	0.049

CM-02

	Feces

	120 h

	49.6526

	56.7563

	7.1037

	64.4761

	14.8235

	0.0433

0.0472 	93.49

108.15	9.08

9.08	84.408

99.068

	1949.365

2098.888	30004.6363  	0.052

CM-02

	Feces

	144 h

	49.9225

	62.9517

	13.0292

	73.9735

	24.0510

	0.0588

0.0563 	60.96

51.89	9.08

9.08	51.878

42.808

	882.270

760.346	19753.2879  	0.034

CM-02

	Feces

	168 h

	49.9487

	61.8468

	11.8981

	72.1372

	22.1885

	0.0562

0.0623 	60.23

63.87	9.08

9.08	51.148

54.788

	910.098

879.414	19853.2934  	0.034

Table 5 (contd).  Recovery of Radioactivity in Feces from Group C.

Subject	Sample	Time	Pot wt	Samp wt	Corr Samp	Homog wt	Corr Homog	Alq wt Orig DPM	Bkg DPM	Calc DPM	LOD	LOQ	DPM/g	Sample DPM	Reco (%)

CM-03

	Feces

	 24 h

	50.0987

	57.2064

	7.1077

	61.1449

	11.0462

	0.0755

0.0688 	3729.77

3194.62	16.42

16.42	3713.352

3178.202

	49183.470

46194.797<PHONE_NUMBER>  	0.850

CM-03

	Feces

	 48 h

	50.7997

	58.1472

	7.3475

	66.0725

	15.2728

	0.0828

0.0773 	964.40

681.73	16.42

16.42	947.982

665.312

	11449.058

8606.882<PHONE_NUMBER>  	0.247

CM-03

	Feces

	 72 h

	49.8413

	56.3469

	6.5056

	64.7264

	14.8851

	0.0633

0.0572 	231.90

231.98	16.42

16.42	215.482

215.562

	3404.139

3768.566	53383.2190  	0.086

CM-03

	Feces

	 96 h

	50.2371

	60.9189

	10.6818

	70.5728

	20.3357

	0.0462

0.0735 	232.63

237.32	9.08

9.08	223.548

228.238

	4838.690

3105.272	80773.0200  	0.130

CM-03

	Feces

	120 h

	50.1252

	56.3624

	6.2372

	64.0774

	13.9522

	0.0568

0.0510 	133.91

120.79	9.08

9.08	124.828

111.708

	2197.667

2190.343	30611.1993  	0.049

CM-03

	Feces

	144 h

	50.7243

	52.3823

	1.6580

	57.2336

	6.5093

	0.0715

0.0905 	98.58

113.16	9.08

9.08	89.498

104.078

	1251.713

1150.028	7816.8261  	0.013

CM-03

	Feces

	168 h

	49.9673

	71.7209

	21.7536

	84.8118

	34.8445

	0.0780

0.0563 	98.24

68.84	9.08

9.08	89.158

59.758

	1143.045

1061.412	38406.6001

	0.062

CM-04

	Feces

	 24 h

	48.8431

	53.9673

	5.1242

	58.0589

	9.2158

	0.0677

0.0766 	2107.54

2386.85	16.42

16.42	2091.122

2370.432

	30888.065

30945.587<PHONE_NUMBER>

	1.861

CM-04

	Feces

	 48 h

	49.6202

	55.5099

	5.8897

	61.3325

	11.7123

	0.0709

0.0863 	749.79

722.30	16.42

16.42	733.372

705.882

	10343.752

8179.397<PHONE_NUMBER>

	0.708

CM-04

	Feces

	 72 h

	49.2083

	56.6738

	7.4655

	65.2357

	16.0274

	0.0846

0.0584 	230.38

187.77	16.42

16.42	213.962

171.352

	2529.102

2934.110	43780.5359

	0.286

CM-04

	Feces

	 96 h

	49.8824

	64.1021

	14.2197

	75.6956

	25.8132

	0.0669

0.0458 	181.88

108.94	9.08

9.08	172.798

99.858

	2582.922

2180.295	61476.9369

	0.401

CM-04

	Feces

	120 h

	49.3234

	56.7203

	7.3969

	66.3387

	17.0153

	0.0470

0.0558 	74.43

87.23	9.08

9.08	65.348

78.148

	1390.372

1400.493	23743.7041

	0.155

CM-04

	Feces

	144 h

	48.8169

	59.0513

	10.2344

	68.8185

	20.0016

	0.0608

0.0514 	59.60

63.18	9.08

9.08	50.518

54.098

	830.880

1052.481	18835.1115

	0.123

CM-04

	Feces

	168 h

	50.5895

	60.6736

	10.0841

	71.0874

	20.4979

	0.0501

0.0520 	50.21

51.19	9.08

9.08	41.128

42.108

	820.908

809.760	16712.6327

	0.109

* in LOD column indicates that sample DPM were less than background DPM, and in LOQ column indicates that sample DPM was less than 3 x background.

Table 6.  Recovery of Radioactivity in CO2 Trap 1 from Group C.

Subject	Sample	Time	Pot wt	Samp wt	Corr Samp	Alq wt	Orig DPM	Bkg DPM Calc DPM	LOD	LOQ	DPM/g	Sample DPM	Reco (%)

CM-01

	CO2 Trap 1

	  1 h

	0.00

	490.30

	490.30

	1.0077

1.0011 	570.60

478.29	18.43

18.43	552.173

459.863

	547.954

459.358<PHONE_NUMBER>

	0.447

CM-01

	CO2 Trap 1

	  3 h

	0.00

	523.66

	523.66

	1.0389

1.0392 	46.46

50.37	29.40

29.40	17.065

20.975

	*

*	16.426

20.184	9585.5499

	0.017

CM-01

	CO2 Trap 1

	  6 h

	0.00

	491.96

	491.96

	1.2911

1.0336 	4759.31

3348.04	15.38

15.38	4743.929

3332.659

	3674.331

3224.322	1696930.6986

	3.073

CM-01

	CO2 Trap 1

	 24 h

	0.00

	500.33

	500.33

	1.0572

1.0435 	14427.83

14047.58	15.38

15.38	14412.449

14032.199

	13632.661

13447.244	6774444.4008

	12.267

CM-01

	CO2 Trap 1

	 48 h

	0.00

	452.14

	452.14

	1.0312

1.0924 	1007.16

1207.98	15.38

15.38	991.779

1192.599

	961.772

1091.724<PHONE_NUMBER>

	0.841

CM-01

	CO2 Trap 1

	 72 h

	0.00

	497.20

	497.20

	1.0415

1.0380 	500.58

490.91	15.38

15.38	485.199

475.529

	465.866

458.121<PHONE_NUMBER>

	0.416

CM-01

	CO2 Trap 1

	 96 h

	0.00

	499.65

	499.65

	1.0526

1.0451 	241.98

220.18	15.38

15.38	226.599

204.799

	215.276

195.961<PHONE_NUMBER>

	0.186

CM-01

	CO2 Trap 1

	120 h

	0.01

	511.23

	511.22

	1.0510

1.0423 	180.24

188.74	15.38

15.38	164.859

173.359

	156.859