# Audited Draft Final Report

ETHYL TERTIARY BUTYL ETHER

ORAL ABSORPTION IN MALE AND

FEMALE RATS

SUBMITTED TO:

Section 211(b) Research Group

American Petroleum Institute

1220 L Street NW

Washington, DC 20005

TESTING FACILITY:

RTI International*

3040 Cornwallis Road

P.O. Box 12194

Research Triangle Park, NC 27709

- 2194

RTI Protocol: RTI

- 93 2

RTI Study Code: Rt05

- 932

RTI Report No. RTI/ 0209408.00 7 and 0212607 .000

February 10 March 6 , 2013

Timothy R. Fennell, Ph.D.

Study Director



### * RTI International is a trade name of Research Triangle Institute.RTI Project No.: 0209408.007 Protocol No. RTI

- 932 Final Report

i SUMMARY

Male and female Fischer 344 rats were administered a single gavage dose of ethyl tertiary butyl ether

(ETBE) in aqueous solution (50 and 10 mg/kg)

. Prior to dosing, the rats were implanted with jugular vein

cannulas for the serial sampling of blood. Blood samples were withdrawn at approximat ely 15, 30 min, 1,

2, 4, 8, and 24 hr. Samples were immediately placed in tared headspace vials and crimp sealed. The

samples were weighed, and after addition of internal standard (MTBE), were analyzed by gas

chromatography coupled with mass spectrometry (GC

- MS) equipped with a headspace autosampler. No

significant levels of ETBE were detected in the predosing samples collected from each animal. ETBE

levels reached maximal values at the first timepoint (0.25 hr) and declined rapidly thereafter, falling to less

that the limit of quantitation by 8 hr at the high dose and 2

- 4 hr at the low dose. Non

- compartmental

pharmacokinetic analysis was conducted for each animal using WinNonlin. No statistically significant

differences between gender were observed in Tmax, apparent Cmax, half life, AUCall,

AUCINF(observed), or lamda z. At the high dose

, elimination half lives of 1.061  0.326 hr for males and

1.105  0.340 hr for females were observed

. At the low dose, eli mination half lives of 0.508  0.191 hr for

males and 0.573  0.176 hr for females were observed. Differences between dose levels in half life and

AUCINF(observed)/Dose may be consistent with saturation of metabolism of ETBE. The study

demonstrated that ETBE is taken up following oral administration, and is rapidly eliminated from blood.RTI Project No.: 0209408.007 Protocol No. RTI

- 932 Final Report

ii Quality Assurance StatementRTI Project No.: 0209408.007 Protocol No. RTI

- 932 Final Report

iii Laboratory GLP Compliance Statement

This study was carried out in compliance with the EPA Good Laboratory Practices (GLP) Standards fo r

Inhalation Exposure Health Effects Testing, 40 CFR part 79, subpart F § 79.60.

The method of synthesis of the test chemical ethyl tertiary butyl ether or its location was not available at

the time of conduct of the study.

There were no significant de viations (Appendix A) that would affect the integrity or quality of the study or

the interpretation of the results. R aw data, and the final report generated as a result of this study are

archived at RTI International.

Timothy R. Fennell, Ph.D. Date

Study DirectorRTI Project No.: 0209 4 08.00 7 Protocol No. RTI

- 932 Final Report

iv Table of Contents

Page

1.0 INTRODUCTION

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

2.0 OBJECTIVES AND PROTO COL

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

3.0 MATERIALS AND METHOD S

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

3.1 Test Substance

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

3.2 Re ference Substances

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

3.3 Test System

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

3.4 Test Chemical Preparation and Analysis

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

3.5 Gavage Dosing

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

3.6 Collection and Analysis of Blood Samples

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

3.6.1 Blood Sample Collection

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

3.6.2 Blood Sample Analysis

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

3.7 Data Collection and Reporting

................................ ................................ ................................ ....... 4 5

3. 8 Pharmacokinetic Analysis

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

4.0 RESULTS

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

4.1 Analysis of Test Chemical Identity and Purity

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

4.2 Method Validation for Analysis of ETBE in Blood

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

4.3 Animal Dosing

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

4.4 Sample Analysis

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

4.5 Pharmacokinetic Analysis

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

5.0 DISCUSSION

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

6.0 REFERENCES

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

7.0 RECORDS AND REPORTS

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

8.0 STORAGE OF RECORDS A ND BIOLOGICAL SAMPLE S

................................ ....................... 9 10

9.0 REGULATORY COMPLIANC E

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

10.0 STUDY PARTICIPANTS

................................ ................................ ................................ ............... 10RTI Project No.: 0209 4 08.00 7 Protocol No. RTI

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v List of Tables

Page

Table 1. Male and Female Fischer 344 Rats Administered ETBE

................................ ....................... 11 12

Table 2. Dose Formulation Analysis.

................................ ................................ ................................ .... 12 13

Table 3. Dose of ETBE Administered by Gavage

................................ ................................ ................ 13 14

Table 4. Individual and Mean Blood Concentrations of ETBE in Male Rats Following Gavage

Administration of 50 mg/kg ETBE.

................................ ................................ ......................... 14 15

Table 5. Individual and Mean Blood Concentrations of ETBE in Female Rats Following Gavage

Administration of 50 mg/kg ETBE.

................................ ................................ ......................... 15 16

Table 6. Individual and Mean Bloods Concentration of ETBE in Male Rats Following Gavage

Administration of 10 mg/kg ETBE.

................................ ................................ ......................... 16 17

Table 7. Individual and Mean Blood Concentrations of ETBE in Female Rats Following Gavage

Administration of 10 mg/kg ETBE.

................................ ................................ ......................... 17 18

Table 8. Pharmacokinetic Analysis of ETBE Following a Single Gavage Dose of ETBE.

................... 18 19

Table 9. Pharmacokinetic Analysis of ETBE Following a Single Gavage Dose of ETBE

(excluding DF05).

................................ ................................ ................................ ................... 19 20

List of Figures

Page

Figure 1. Headspace GC

- MS Analysis of ETBE in control blood with no added internal standard.

...... 20 21

Figure 2. Headspace GC

- MS Analysis of ETBE in control blood with internal standard.

...................... 20 21

Figure 3. Headspace GC

- MS Analysis of ETBE in predose blood with internal standard.

................... 20 22

Figure 4. Headspace GC

- MS Analysis of ETBE in rat blood following dose administration.

................ 21 22

Figure 5. Example pharmacokinetic analysis for blood concentration of ETBE for Animal AM01,

administered 50 mg/kg ETBE by gavage.

................................ ................................ ............. 22 23

List of Appendices

Appendix A: Approved Study Protocol, Amendments, and Deviations

Appendix B: Test Chemical Analysis Report Ethyl Tertiary Butyl Ether l RTI Reference 12307

- 11

Appendix C: Method Validation Plan, Analytical Method and Validation Report

Appendix D: Pharmacokinetic Analysis

Appendix E: Dose Formulation Analysis

Appendix F: Blood Sample Collection and Analysis for ETBERTI Project No.: 0209 4 08.00 7 Protocol No. RTI

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viRTI Project No.: 0209 4 08.00 7 Protocol No. RTI

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1 1.0 INTRODUCTION

Ethyl tertiary butyl ether (ETBE) is an oxygenate used in the manufacture of blended gasolines.

A number of studies have investigated the metabolism and pharmacokinetics of ETBE following inhalation

exposure in rodents and humans (Amberg et al., 2000; Bernauer et al ., 1998; Dekant et al

., 2001;

Johanson et al., 1995; Nihlen et al ., 1998a, 1998b) . Metabolism of ETBE occurs via oxidation and

subsequent conjugation or further oxidation (Bernauer et al. 1998; Dekant et al. 2001) . Physiologically

based pharmacokinetic models have been developed to describe the behavior of ETBE in rodents and

humans. Despite intensive investigations of the behavior of ETBE following inhalation exposure, the

kinetics of uptake and elimination following oral administration in rats had not been investigated

previously

.

2.0 OBJECTIVES AND PROTO COL

The objectives of this study on ETBE were to:

Develop a method for the analysis of ETBE in blood by headspace gas chromatography

– mass

spectrometry (GC

- MS).

Conduct an evaluation of oral absorption by measuring ETBE in blood in male and female Fischer

344 rats administered ETBE by gavage at one of two dose levels, 10 and 50 mg/kg.

Copies of the approved protocol and protocol amendments for these studies are included in

Appendix A.

3.0 M ATERIALS AND METHODS

3.1 Test Substance

NAME : Ethyl tertiary Butyl Ether (ETBE; tertiary butyl ethyl ether, 2

- ethoxy

- 2

- methylpropane)

CAS No.: 637

- 92

- 3

MOLECULAR FORMULA : C 6 H 14 O

MOLECULAR WEIGHT : 102.17

STRUCTURE:

SOURCE OF TEST SUBSTANCE: ETBE was purchased from Sigma

- Aldrich, Milwaukee, WI (Catalog

number 253898, specified purity 99%). A certificate of analysis was obtained from the vendor

,

which indicated a purity of 99.3%

.RTI Project No.: 0209 4 08.00 7 Protocol No. RTI

- 932 Final Report

2 LOT NUMBER(S): 04608LD

IDENTITY AND PURITY: The identity of the unlabeled ETBE was confirmed at RTI by 1 H and 13 C NMR,

and by mass spectrometry. Details of the analysis conducted are contained in Appendix C. The

purity of the test chemical was determined by GC with flame ionization detec tion (FID) also

contained in Appendix B

.

STORAGE CONDITIONS : ETBE was stored in the dark at room temperature.

STABILITY

: Upon receipt, the identity and purity of the test substance ETBE was confirmed as described

above. Periodically, and at the end of the experimental portion of the study, ETBE was reanalyzed

by 1 H NMR and by GC

- MS to verify stability. Initial analysis in March 2006 indicated a purity of 99.66

 0. 0 4 %, with purity following the completion of the study of 99. 26  0. 17 %. Details of the analysis

are included in Appendix C.

3.2 Reference Substances

Methyl tertiary butyl ether (MTBE) was obtained from Sigma Aldrich ( Milwaukee, Wi, Catalog

number 443808, specified purity 99 % ) . This material was assigned a chemical receipt number of BOC

- B

0456

.

3. 3 Test System

Source: M ale Fischer 344 rats for methods development were purchased from Charles River

( Kingston , NY). Male and female Fischer 344 rats with jugular vein cannula e were obtained from Harlan

Sprague Daw le y (Indianapolis, In).

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 per formed 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 Dur ham, NC. Approximately once a year, the City of Durham provides

analyses of the drinking water for potential contaminants. Documentation of these analyses were

inspected by the Study Director and maintained in the study records. In addition, samples of water were

collected during the study for analysis of tertiary butyl alcohol, 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 analy sis.

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 expe riment. 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 were housed individually in polycarbonate cagesRTI Project No.: 0209 4 08.00 7 Protocol No. RTI

- 932 Final Report

3 with dimensions of 9 1⁄4” x 8 1⁄4” x 8” (ca. 76.3 sq. in. floor space) , and were used within 1

- 3 days of arrival

at RTI.

Following dosing, the cannulated animals were housed individually in polycarbonate cages with

dimensions of 9 1⁄4” x 8 1⁄4” x 8” (ca. 76.3 sq. in. floor space)

, and were placed in a hood

.

Quarantine: Rats without cannulas that were used as blood donors were quarantined for a

minimum of one week. Rats with implanted jugular vein cannulas received a truncated quarantine period

of 1 day. A veterinarian or qualifie d 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 gen erated numbers as described in SOP DPK

- HUS

- 001 Assi gnment 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 ), Study B

(n= 9 ), Study C (n= 9 ), and Study D (n= 9 ). Ani mals were ordered specifically for each study, with Studies

C and D performed 1 week later than Studies A and B. Of the animals in each group, the first four

animals were administered ETBE and the remaining animals were held as extras in the event of dosi ng

accidents or inability to obtain blood samples.

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 Revisio n 4.4.1 for Signal  software [Siebe Environmental Controls (SEC)/Barber

- Colman

Company; Loves Park, IL]). The target environmental ranges were 6 4

 7 9  F ( 18  C

- 2 6  C) for

temperature and 3 0

 70% relative humidity, with a 12

- h light cycle per day.

Euthanas ia: At the end of the in

- life phase, the rats were euthanized by overexposure to carbon

dioxide.

3. 4 Test Chemical Preparation and Analysis

ETBE was prepared for gavage dosing as a solution in water

. For dosing of animals, the dose

solution was prepared within 24 hours of dosing, and maintained in a refrigerator

. The nominal

concentration was calculated from the weight of ETBE and the weight of water added to the dose

preparation. Dose solutions were prepared at two concentrations, the first a t approximately 5 mg/ml and

the second at 1 mg/ml , to ensure that the volume of dose administered per kg body weight is similar in

the high and the low dose groups. Prior to dosing, a method was developed for the quantitative analysis

of ETBE in water, by dissolving a weighed aliquot of the ETBE/water solution in methanol (see Appendix

C ) . Aliquots of this solution were analyzed directly by gas chromatography (GC) using a standard curve

for the quantitation of ETBE. The stability of ETBE in water was de termined by analysis of aliquots of a

mock dose solution of ETBE kept at room temperature for three days (Appendix E)

.RTI Project No.: 0209 4 08.00 7 Protocol No. RTI

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4 3. 5 Gavage Dosing

Each rat was weighed prior to dosing to determine the amount of dose to be administered. A

single gavage dose was administered using a syringe fitted with a blunt tipped gavage dosing needle.

The dose administered to each animal was determined from the weight of the full syringe minus that of

the empty syringe. The dose time was recorded. Dosing of animals was spa ced apart to allow blood

collection at the appropriate times (see section 3

. 6 .1 below). The groups were designated according to

the dose administered and sex of the rats: Group A male 50 mg/kg, Group B female 50 mg/kg, Group C

male 10 mg/kg, and Group D f emale 10 mg/kg. At least four cannulated rats per group were dosed with

ETBE. Group and animal numbers are indicated in Table 1.

3. 6 Collection and Analysis of Blood S amples

3. 6 .1 Blood Sample Collection

Blood samples were collected prior to dosing fro m each rat (time = 0), from 4 rats of each sex, at

each dose at approximately 15 and 30 min, and at approximately 1, 2, 4, 8, and 24 hr after dosing.

Blood samples (approximately 100 μ l) were drawn from the jugular vein cannula into a

heparinized 1 ml syringe, and the sample was immediately placed in a preweighed headspace vial. The

vial was immediately crimped and weighed, and the weight of the blood aliquot was determined. After the

last s ample had been collected from each animal at 24 hr, the rats were euthanized by exposure to CO 2

.

When cannulas fail ed during the collection of samples, several samples were collected at 15 and 30 min,

and at 8 hr

, via the tail vein, and at 24 hr, blood was collected by cardiac puncture under CO 2 anesthesia

at sacrifice. The blood samples were maintained on ice, until placed in the headspace autosampler for

analy sis

. All blood samples were analyzed by GC

- MS within 24 hours of collection.

Blood samples fr om unexposed rats were used for method development. Blood samples were

collected by cardiac puncture, under CO 2 anesthesia, and the rats were euthanized.

3. 6 .2 Blood Sample Analysis

A GC

- MS method was developed for quantitating ETBE using blood from cont rol male Fischer

344 rats. The methods involve d the development of a standard curve for analysis of ETBE in the

headspace of airtight vials containing a known volume of cont r ol blood and a known concentration of

ETBE. The stability of ETBE was evaluated under storage conditions that were used in this study. This

include d analysis of ETBE immediately after spiking in control blood, after storage at room temperature (8

hr), or at approximately 4 o C (24 hr). The concentration of ETBE in the heads pace of vials containing a

known volume of blood from rats administered ETBE by gavage was determined by comparison to the

standard curve.

3. 7 Data Collection and Reporting

Study data was coll ected and reported in the Debra TM s ystem version 5.5.10. 7 5 (L ablogic

Systems Limited, Sheffield, England) . This includes data for dosing, dose times, sample collection times,RTI Project No.: 0209 4 08.00 7 Protocol No. RTI

- 932 Final Report

5 and pot weights and sample weights. Calculation s of sample data are described in Appendix G , and

were reported with the Debra TM s ystem

.

3.7 Pharmacokinetic Analysis

Individual and mean blood concentration

- time data for each dose and sex were analyzed by

noncompartmental (model

- independent) methods using the least

- squares fitting program WinNonlin 

(Statistical Consulting Inc., Cary, NC). P harmacokinetic parameters were extracted from the WinNonlin

generated outputs. These include:

Pharmacokinetic Parameters

WinNonlin Output Table/Report

Tmax T

max Time of maximum observed concentration.

Cmax C

max Maximum observed concentration, occurring at

T

max

lambda_z 

z Terminal elimination rate constant

t1/2_lamda_z T 1/2  z Terminal elimination half

- life

AUCall AUCall Area under the plasma concentration

- time curve

from time zero to the last measured time point

AUCINF(observed) AUCINF(observed) Area under the plasma concentration

- time curve

from time zero extrapolated to infinity

AUCINF(observed)/D AUCINF(observed)/

Dose Area under the plasma concentration

- time curve

from time zero extrapolated to infinity/Dose

Vz(observed)/F* Vz(observed)/F Volume of Distribution at steady state

Cl(observed)/F* Clearance(observed)/F Systemic clearance

MRTlast MRTlast Mean residence time based upon values from the

time of dosing up to the last measured

concentration

*For extravascular models the fraction of dose absorbed cannot be estimated, therefore Volume

of Distribution and Clearance for these models are actually Volume/F or Clearance/F where F is

the fraction of dose absorbed.

Statistical comparison of the parameters Tmax, Cmax, t1/2 La mbda z, Lambda z, AUCall,

AUCobserved and AUCobserved/Dose were conducted using the Student’s t

- test available in SAS®

Version 9 (SAS Institute Inc., 2004, 2005, 2006a,b). The males were compared to the females within

dose group for each parameter. Additi onally, for the parameter AUCobserved/Dose the low dose was

compared to the high dose within each sex. Grubbs test for outliers was conducted to determine whether

individual values were outliers (Grubbs, 1969)

.RTI Project No.: 0209 4 08.00 7 Protocol No. RTI

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6 4.0 RESULTS

4.1 Analysis of 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. 66  0. 04 %. Following

completion of the stu dy, test chemical purity was determined to be 99. 26  0. 17 % , indicating that the test

chemical was stable under the conditions of storage

.

4.2 Method Validation for Analysis of ETBE in Blood

A method for the determination of ETBE in blood using headsp ace GC

- MS was developed and

validated prior to the study conduct. Details of the method, the method validation plan, and the validation

report are contained in Appendix C. The analytical method titled Project Specific Method for Analysis of

Ethyl tertiar y Butyl Ether in Blood Samples (AM

- 0209408.007) was validated in preparation for analyses

of blood samples to determine concentrations of e thyl tertiary butyl ether (ETBE) present in blood in this

study . This validation established a Limit of Quantitation (LOQ), accuracy, and precision of the method.

Th e method consists of GC/MS analysis of ETBE with injection of headspace from sample vials.

The validation procedure established a calibration range of 0.110  g/ml to 250  g/ml for analysis

of ETBE in the headspace of blood samples using two calibration curves. The low

- concentration curve

was defined by a linear regression (y=bx+a) of slope 0.08065, intercept

- 0.0006558, and linear correlation

coefficient of 0.9998. The high

- concentration curve was define d by the linear regression (y=bx+a) of

slope 0. 09673 , intercept

- 0.1292, and linear correlation coefficient of 0.9994 . Using the regression

equation, calculated concentrations for all calibration standards were within the acceptance criteria of ±

15% (20% for the LOQ) of the nominal concentrations. The limit of quantitation was established as 0.11

 g/ml. Mean calculated concentrations for the replicate concentration points assayed at 0.540  g/ml,

5.06  g/ml, and 101  g/ml for determination of precision a nd accuracy ranged from 96.2% to 99.3% of

nominal concentration. Precision around the mean for each of these replicate concentration points was

calculated to be well within the acceptance criteria of 15% CV. Additionally, the mean values for each

concent ration were well within the acceptance criteria of 15% deviation from nominal concentration.

Storage stability of ETBE in blood was verified at room temperature for 8 h r , and at approximately 4 o C for

24 h r . Storage stability of standard solutions was v erified at 7 days at approximately

- 20 o C.

4. 3 Animal Dosing

Animals were administered a single gavag e dose of ETBE in water. Analysis of the ETBE dose

solutions was conducted prior to dosing by GC with FID detection (see Table 2 ). The concentration of

ETBE determined analytically was used to calculate the amount of ETBE dose solution to administer.

The amount of dose solution administered was calculated by the difference between the weight of the

charged syringe and needle with dose, and the discharge d syringe and needle after dosing. Information

on the doses administered is reported in Table 3. The doses administered were within  10% of theRTI Project No.: 0209 4 08.00 7 Protocol No. RTI

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7 target dose. Doses of ETBE were administered to 4 animals in each group AM, BF, and DF. Five

animals were d osed in group CM, but one of these (CM

- 04) was dosed with less than the expected dose

per kg body weight due to a dosing error (16.6 % less than expected) . The error was discovered during

the review of the dosing data immediately following dosing. One of the extra animals (the next animal in

the series that had a patent cannula, CM

- 08) was dosed, and the samples that had been obtained from

rat CM

- 04 were not analyzed with the exception of the predose sample, which was in the process of

analysis

.

4.4 Sample Analysis

Sample analysis was conducted on the day of collection, with a target of completing sample

analysis within 16 hr of sample collection. Calibration curves prepared with rat blood with known

concentrations of ETBE added were constructed, at the same time as sample analysis. Linear regression

equations were derived for two standard curves: a low concentration curve, with concentrations ranging

from 0.1  g/ml to 5.2  g/ml; and a high concentration curve, with concentrations ranging from 5.2 to 250

 g/ml. Details of the calibration curves and method performance are provided in Appendix F . Blood

concentration data for each dose group are reported in Tables 5

- 10

.

With gavage administration of 50 mg ETBE/kg, ETBE reach ed a maximum in blood at t he first

timepoint (0.25 h)

, and declined rapidly, falling to <LOQ at 8 h r (Tables 7 ). Similarly, with gavage

administration of 10 mg ETBE/kg, ETBE reach ed a maximum in blood at the first timepoint (0.25 h), and

declined rapidly, falling to <LOQ by 2

- 4 hr in males and 2

- 4 hr in females (Tables 10 ).

4. 5 Pharmacokinetic Analysis

Pharmacokinetic analysis was conduc t ed using a non

- compartmental model for extravascular

input with uniform weighting. The WinNonlin output reports for individual animals are included in

Appendix D. An example semilog plot of ETBE concentration vs. time for rat BF01 is shown in Figure 5.

Semilog plots for each of the animals are shown in Appendix D. For animal DF05, the pharmacokinetic

behavior was considered different from the other animals in the group, and this animal was treated as a

pharmacokinetic outlier as per the study protocol, section 9.0 Pharmacokinetic Analysis, item 4. A

Grubbs tes t for outlier was not significant for the concentration of ETBE at 0.25 h. Data including and

excluding this animal are presented in Tables 8 and 9. With DF05 included, the calculated half life for the

low dose female group (DF) was 1.558  1.976 hr (mea n  standard deviation ). However, a Grubbs test

for outlier indicated that the value of t1/2_lambda_z of 4.513 h for DF05 was an outlier when compared

with the mean value of 1.558 and standard deviation of 1.976 h for the DF group (p<0.05). Exclusion of

DF05 reduced the mean half life by a factor of approximately three to 0.573  0.176 hr (Ta b le 9)

.

The mean Tmax was achieved at the first time point following dosing in all dose groups (Table 9).

This precluded accurate determination of Cmax and Tmax, s ince the absorption phase was not captured

in the data obtained. The apparent Cmax values measured for males and females in the high dose

groups, (AM and BF) were similar at 6.514  0.727 , and 5.837  1.586  g/ml. In the low dose groups, theRTI Project No.: 0209 4 08.00 7 Protocol No. RTI

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8 Cmax values measured for males and females ( C M and D F) were similar at 1.057  0.174

, and 1.103 

0.133  g/ml. The half lives in males and females at the high dose were similar ( 1.061  0.326 hr for

males and 1.105  0.340 hr for females). While there were no significant gender differences, the half

lives in males and females at the low dose were approximately half of those observed at the high dose

( 0.508  0.191 h for males and 0.573  0.176 h for females)

. There was little difference in AUCall and

AUCINF(obs erved) between genders, at both the high and low doses. However, when

AUCINF(observed) was normalized by dose (AUCINF(observed)/D), the values calculated for the high

dose ( 0.123  0.014  g.h/ml/mg/kg for AM, and 0.122  0.004 for BF compared with 0.066  0.004 for CM

and 0.078  0.016 for DF ) were significantly different from the low dose

.

5.0 DISCUSSION

This investigation indicated that the uptake of ETBE was rapid following gavage administration

,

with the highest concentrations observed at the fir st timepoint, and therefore the apparent Cmax was

achieved at the first timepoint. No information on the uptake rate was determined from pharmacokinetic

modeling, because the uptake phase was not captured in the sampling schedule. Since the absorption

pha se of the kinetic curve was not captured, the maximal concentrations of ETBE achieved may have

occurred at a time earlier than the first sample, and may have been higher than that observed. ETBE

was rapidly eliminated, and fell below the LOQ by 8 hr at th e high dose, and by 4 hr in the low dose

males, and 2

- 4 hr in the low dose females.

No statistically significant

. differences between males and females were observed in the

pharmacokinetic parameters measured. There were however differences between the hi gh and the low

dose groups that were similar in males and females. The longer half lives and increased

AUCINF(observed)/D at the high dose are consistent with saturation of metabolism, which may be

expected based on the requirement for oxidative metabolis m as one of the mechanisms for elimination of

ETBE. However, because the concentration of ETBE rapidly reached LOQ at the low dose, the ability to

determine saturation is limited.

6.0 REFERENCES

Amberg, A., Rosner, E. and Dekant, W . (2000). Biotransformation and kinetics of excretion of ethyl tert

butyl ether in rats and humans. Toxicol Sci 53:194

- 201.

Bernauer, U., Amberg, A., Scheutzow, D. and Dekant, W. (1998). Biotransformation of 12C

- and 2

- 13C

labeled methyl tert

- butyl ether, ethyl tert

- butyl ether, and tert

- butyl alcohol in rats: identification of

metabolites in urine by 13 C nuclear magnetic resonance and gas chromatography/mass spectrometry.

Chem Res Toxicol 11:651

- 8.RTI Project No.: 0209 4 08.00 7 Protocol No. RTI

- 932 Final Report

9 Dekant, W., Bernauer, U., Rosner, E. and Amberg, A. (2001). Biotransformation of MTBE, ETBE, and

TAME after inhalation or ingestion in rats and humans. Res Rep Health Eff Inst:29

- 71; discussion 95

- 109.

Grubbs, F.E. (1969) Procedure for detecting outlying observations in samples. Technometrics, 11: 1

- 21.

Johanson, G., Nihlen, A. and Lof, A. (1995). Toxicokinetics and acute effects of MTBE and ETBE in male

volunteers. Toxicol Lett 82

- 83:713

- 8.

Nihlen, A., Lof, A. and Johanson, G. (1998a). Controlled ethyl tert

- butyl ether (ETBE) exposure of male

volunteers . I. Toxicokinetics. Toxicol Sci 46:1

- 10.

Nihlen, A., Lof, A. and Johanson, G. (1998b). Controlled ethyl tert

- butyl ether (ETBE) exposure of male

volunteers. II. Acute effects. Toxicol Sci 46:143

- 50.

SAS Institute Inc. (2004). SAS/STAT® User’s Guide, Versi on 9.1, Cary, NC: 5136 pp.

SAS Institute Inc. (2005). SAS® Language Reference: Concepts, Version 9.1.3, Cary, NC: 664 pp.

SAS Institute Inc. (2006a). SAS® Language Reference: Dictionary, Version 9.1.3, Cary, NC: 1908 pp.

SAS Institute Inc. (2006b). SAS® Procedures Guide, Version 9.1.3, Cary, NC: 1934 pp.

7.0 RECORDS AND REPORTS

The following will be maintained in the record:

a) Protocol and any amendments

b) Animal receipt records

c) Quarantine records

d) Temperature and humid ity records for the treatment rooms

e) Animal research facility room logs

f) Feed and water analysis for contaminants

g) Randomization records

h) Test chemical receipt, storage, and use records

i) Balance calibration log references

j) Correspondences

k) All other raw data and documentation

Upon acceptance of the audited draft report by the Sponsor, a final report will be issued.

8.0 STORAGE OF RECORDS A ND BIOLOGICAL SAMPLE S

A copy of the final report and the records for this study, including all raw data, will be retained i n

the RTI Archives, under the responsibility of the RTI Quality Assurance Unit, for the length of time

specified in the appropriate regulations.RTI Project No.: 0209 4 08.00 7 Protocol No. RTI

- 932 Final Report

10 The test substance ETBE is an ether that can generate explosive peroxides on storage.

Therefore, an archived s ample of test chemical will not be retained. The samples of blood collected will

not afford analysis beyond the duration of the study. Wet specimens of blood will be disposed of after

quality assurance verification (after the QAU assures that discarding the samples does not negatively

impact the integrity of the study)

.

Materials will be maintained in the RTI Archive for a period of one year after the signature of the

final report as part of the initial study cost. At that point, the Sponsor will be contacted to determine the

final disposition of these materials. The Sp onsor may continue to store these materials in the RTI

Archive, have RTI ship them to the Sponsor or an alternative archive facility, or have RTI dispose of them.

The Sponsor will be responsible for all costs associated with the storage of these materials beyond 1 year

from the issuance of the final report, and for any costs associated with the shipment of these materials to

the Sponsor or to any other facility designated by the Sponsor. The Sponsor will be notified in writing

when the RTI retention time h as expired.

9.0 REGULATORY COMPLIANC E

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: Christopher Johnson , D.V.M.

Serena Young, D.V.M

Animal Facility Supervisor: Natal ie Ostin

Laboratory Staff: N orman F. Gaudette, BS

Y an Hong, BS

M elody . P. Gower

P urvi . Patel

Donna Coleman

Scott Watson

Jennifer Demeter

Christine Myers

Secretary: K athleen . G. AnchetaRTI Project No.: 0209 4 08.00 7 Protocol No. RTI

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11 Table 1

. M ale and Female Fischer 344 Rats Administered ETBE

Group Subject Species Strain Gender Timepoint Body Weight a

(g)

Group A AM01 Rat Fischer 344 Male 0 m 150.57

Group A AM02 Rat Fischer 344 Male 0 m 161.91

Group A AM03 Rat Fischer 344 Male 0 m 158.09

Group A AM04 Rat Fischer 344 Male 0 m 146.05

Group B BF01 Rat Fischer 344 Female 0 m 135.82

Group B BF02 Rat Fischer 344 Female 0 m 132.95

Group B BF03 Rat Fischer 344 Female 0 m 138.13

Group B BF04 Rat Fischer 344 Female 0 m 131.39

Group C CM01 Rat Fischer 344 Male 0 m 139.26

Group C CM02 Rat Fischer 344 Male 0 m 141.44

Group C CM03 Rat Fischer 344 Male 0 m 135.27

Group C CM04 Rat Fischer 344 Male 0 m 180.64

Group C CM08 Rat Fischer 344 Male 0 m 150.54

Group D DF01 Rat Fischer 344 Female 0 m 136.36

Group D DF02 Rat Fischer 344 Female 0 m 129.57

Group D DF03 Rat Fischer 344 Female 0 m 140.44

Group D DF04 Rat Fischer 344 Female 0 m 137.40

Group D DF05 Rat Fischer 344 Female 0 m 130.57

a Body weight on the day of dosingRTI Project No.: 0209 4 08.00 7 Protocol No. RTI

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12 Table 2

. Dose Formulation Analysis

.

Dose Formulation Nominal

Concentration

( mg/ml ) Actual Concentration ( mg/ml )

Mean SD

ETBE 5 mg/mL Group A 5 5.2 388 0.096 0

ETBE 5 mg/ml Group B 5 5. 2859 0. 0707

ETBE 1 mg/mL Group C 1 1.1486 0.0058

ETBE 1 mg/mL Group D 1 0.9902 0.0081RTI Project No.: 0209 4 08.00 7 Protocol No. RTI

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13 Table 3

. Dose of ETBE Administered by Gavage

Group Subject Treatment Full

Syringe

Weight

( g) Empty

Syringe

Weight

(g) Dose

Solution

Admin.

(g) ETBE

Admin.

(mg) Actual

Dose

(mg/kg) Dose

error

(%)

A AM01 ETBE 5 mg/mL

Group A 7.7369 6.3137 1.4232 7.4559 49.518

- 0.96 %

A AM02 ETBE 5 mg/mL

Group A 7.8845 6.3096 1.5749 8.2506 51.958 1.92 %

A AM03 ETBE 5 mg/mL

Group A 7.8140 6.3036 1.5104 7.9127 50.052 0.10 %

A AM04 ETBE 5 mg/mL

Group A 7.6482 6.3065 1.3417 7.0289 48.127

- 3.75 %

B BF01 ETBE 5 mg/ml

Group B 7.2363 5.9442 1.2921 6.8299 50.286 0.57 %

B BF02 ETBE 5 mg/ml

Group B 7.2166 5.9473 1.2693 6.7094 50.466 0.93 %

B BF03 ETBE 5 mg/ml

Group B 7.2905 5.9456 1.3449 7.1090 51.466 2.93 %

B BF04 ETBE 5 mg/ml

Group B 7.2328 5.9409 1.2919 6.8289 51.974 3.95 %

C CM01 ETBE 1 mg/mL

Group C 7.5584 6.3083 1.2501 1.4359 10.311 3.11 %

C CM02 ETBE 1 mg/mL

Group C 7.6016 6.3055 1.2961 1.4887 10.525 5.25 %

C CM03 ETBE 1 mg/mL

Group C 7.4832 6.2976 1.1856 1.3618 10.067 0.67 %

C CM04 a ETBE 1 mg/mL

Group C 7.6203 6.3091 1.3112 1.5060 8.337

- 16.63 %

C CM08 ETBE 1 mg/mL

Group C 7.6597 6.3382 1.3215 1.5179 10.083 0.83 %

D DF01 ETBE 1 mg/mL

Group D 7.3260 5.9315 1.3945 1.3808 10.126 1.26 %

D DF02 ETBE 1 mg/mL

Group D 7.2674 5.9193 1.3481 1.3349 10.302 3.02 %

D DF03 b ETBE 1 mg/mL

Group D 7.3515 5.9046 1.4469 1.4327 10.202 2.02 %

D DF04 ETBE 1 mg/mL

Group D 7.3276 5.9231 1.4045 1.3907 10.122 1.22 %

D DF05 ETBE 1 mg/mL

Group D 7.2577 5.9076 1.3501 1.3369 10.239 2.39 %

a Rat CM04 was removed from the study because of a dosing error.

b Rat DF03 was removed from the study because of loss of cannula patency.RTI Project No.: 0209 4 08.00 7 Protocol No. RTI

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14 Table 4

. Individual and Mean Blood Concentration s of ETBE in Male Rats Following Gavage

Administration of 50 mg/kg ETBE.

ETBE Concentration (  g/ml)

Time

(h) AM01 AM02 AM03 AM04 Mean SD

0 <LOQ a <LOQ <LOQ <LOQ <LOQ <LOQ

0.25 5.91 5.95 7.44 6.76 6.51 0.727

0.5 3.59 3.04 4.99 2.97 3.65 0.938

1 1.73 1.88 2.36 1.38 1.84 0.405

2 0.566 0.630 0.690 0.673 0.640 0.0553

4 0.311 0.269 0.261 0.336 0.294 0.0356

8 <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ

24 <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ

a Less than the limit of quantitation, 0.11  g/mlRTI Project No.: 0209 4 08.00 7 Protocol No. RTI

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15 Table 5

. Individual and Mean Blood Concentration s of ETBE in Female Rats Following Gavage

Administration of 50 mg/kg ETBE.

ETBE Concentration (  g/ml)

Time

(h) BF01 BF02 BF03 BF04 Mean SD

0 <LOQ a <LOQ <LOQ <LOQ <LOQ <LOQ

0.25 5.16 4.15 6.16 7.87 5.84 1.59

0.5 4.12 2.92 3.33 4.05 3.60 0.578

1 1.95 2.21 1.50 1.98 1.91 0.298

2 1.10 0.923 0.697 0.627 0.836 0.215

4 0.197 0.306 0.347 0.193 0.261 0.0779

8 <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ

24 <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ

a Less than the limit of quantitation, 0.11  g/mlRTI Project No.: 0209 4 08.00 7 Protocol No. RTI

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16 Table 6

. Individual and Mean Blood s Concentration of ETBE in Male Rats Following Gavage

Administration of 10 mg/kg ETBE.

ETBE Concentration (  g/ml)

Time

(h) CM01 CM02 CM03 CM0 8 Mean SD

0 <LOQ a <LOQ <LOQ <LOQ <LOQ <LOQ

0.25 1.02 1.24 1.14 0.83 4 1.06 0.1 7

0.5 0.42 3 0.50 3 0.5 47 0.4 36 0.48 0.05

1 0.219 0.221 0.270 0.190 0.225 0.0 33

2 0.09 58 <LOQ <LOQ 0.080 0 0.08 79 0.0 122

4 0.029 <LOQ <LOQ <LOQ 0.029 0.000

8 <LOQ <LOQ <LOQ < LOQ <LOQ <LOQ

24 <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ

a Less than the limit of quantitation, 0.11  g/mlRTI Project No.: 0209 4 08.00 7 Protocol No. RTI

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17 Table 7

. Individual and Mean Blood Concentration s of ETBE in Female Rats Following Gavage

Administration of 10 mg/kg ETBE.

ETBE Concentration (  g/ml)

Time

(h) DF01 DF02 DF0 4 DF0 5 Mean SD

0 <LOQ a <LOQ <LOQ <LOQ <LOQ <LOQ

0.25 1.26 1.05 1.01 0.282 0.898 0.425

0.5 0.653 0.554 0.460 0.308 0.494 0.147

1 0.320 0.259 0.245 0.269 0.273 0.033

2 0.120 0.095 0.101 <LOQ 0.110 0.014

4 <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ

8 <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ

24 <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ

a Less than the limit of quantitation, 0.11  g/mlRTI Project No.: 0209 4 08.00 7 Protocol No. RTI

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18 Table 8

. Pharmacokinetic Analysis of ETBE Following a Single Gavage Dose of ETBE.

Parameter AM a

Male Rats

50 mg/kg BF a

Fema le Rats

5 0 mg/kg CM a

Male Rats

1 0 mg/kg DF b

Fema le Rats

1 0 mg/kg

(units) Mean SD Mean SD Mean SD Mean SD

Tmax (h) 0.243 0.012 0.235 0.037 0.247 0.018 0.316 0.118

Cmax (  g/ml) 6.514 0.727 5.837 1.586 1.057 0.174 0.904 0.412

t1/2_Lambda_z (h) 1.061 0.326 1.105 0.340 0.508 0.191 1.558 1.976

Lambda_z (1/h) 0.694 0.181 0.667 0.174 1.525 0.581 1.018 0.691

AUCall (  g.h/ml) 5.641 0.762 5.795 0.401 0.571 0.035 0.560 0.268

AUCINF(observed)

(  g.h/ml) 6.102 0.670 6.237 0.247 0.675 0.043 1.085 0.600

AUCINF(observed)/D

(  g.h/ml/mg/kg) 0.123 0.014 0.122 0.004 0.066 b 0.004 0.106 c 0.058

a Values represent mean and standard deviation, n = 4.

b Significantly different from AM, 50 mg/kg, p<0.05

c Significantly different from BF, 50 mg/kg, p<0.05RTI Project No.: 0209 4 08.00 7 Protocol No. RTI

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19 Table 9

. Pharmacokinetic Analysis of ETBE Following a Single Gavage Dose of ETBE (excluding

DF05).

Parameter AM a

Male Rats

50 mg/kg BF a

Fema le Rats

5 0 mg/kg CM a

Male Rats

1 0 mg/kg DF b

Fema le Rats

1 0 mg/kg

(units) Mean SD Mean SD Mean SD Mean SD

Tmax (h) 0.243 0.012 0.235 0.037 0.247 0.018 0.257 0.023

Cmax (  g/ml) 6.514 0.727 5.837 1.586 1.057 0.174 1.103 0.133

t1/2_Lambda_z (h) 1.061 0.326 1.105 0.340 0.508 0.191 0.573 0.176

Lambda_z (1/h) 0.694 0.181 0.667 0.174 1.525 0.581 1.306 0.468

AUCall (  g.h/ml) 5.641 0.762 5.795 0.401 0.571 0.035 0.674 0.170

AUCINF(observed)

(  g.h/ml) 6.102 0.670 6.237 0.247 0.675 0.043 0.792 0.156

AUCINF(observed)/D

(  g.h/ml/mg/kg) 0.123 0.014 0.122 0.004 0.066 c 0.004 0.078 d 0.016

a Values represent mean and standard deviation, n = 4.

b Values represent mean and standard deviation, n = 3

.

c Significantly different from AM, 50 mg/kg, p<0.05

d Significantly different from BF, 50 mg/kg, p<0.05RTI Project No.: 0209 4 08.00 7 Protocol No. RTI

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20 Figure 1

. Headspace GC

- MS Analysis of ETBE in control blood with no added internal standard.

Upper panel: selected ion monitoring at m/z 87 (ETBE), and lower panel, selected ion monitoring at m/z

73 (MTBE, internal standard).

Figure 2

. Headspace GC

- MS Analysis of ETBE in control blood with internal standard.

Upper panel: selected ion monitoring at m/z 87 (ETBE), and lower panel, selected ion monitoring at m/z

73 (MTBE, internal standard).

Figure 3

. Headsp ace GC

- MS Analysis of ETBE in predose blood with internal standard.RTI Project No.: 0209 4 08.00 7 Protocol No. RTI

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21 Upper panel: selected ion monitoring at m/z 87 (ETBE), and lower panel, selected ion monitoring at m/z

73 (MTBE, internal standard).

Figure 4

. Headspace GC

- MS Analysis of ETBE in rat blood following dose administration.

Upper panel: selected ion monitoring at m/z 87 (ETBE), and lower panel, selected ion monitoring at m/z

73 (MTBE, internal standard).RTI Project No.: 0209 4 08.00 7 Protocol No. RTI

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22 Figure 5

. Example pharmacokineti c analysis for blood concentration of ETBE for Animal BF 01

,

administered 50 mg/kg ETBE by gavage

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Approved Study Protocol, Amendments, and DeviationsRTI Project No.: 0209 4 08.00 7 Protocol No. RTI

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- 22 RTI Protocol No. RTI

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Deviations from the Approved Protocol

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

Deviation

The protocol states (section 8.5.1) that: “The blood samples will be maintained at room temperature until

analyzed.”

Reason for Deviation

Oversight. The storage conditions to be investigated and found to maintain ETBE concentrations were

noted in section 8.6: "This will include analysis of ETBE immediately after spiking in control blood, after

storage at room (8 hr), or at approximately 4 o C (24 hr)."

Deviation

The protocol states (section 7.1.5) that:

“Following administration of ETBE, the animals will be placed in the hood for the duration of the post

dosing period.” Group C animals dosed the previous night were transferred out of the hood in the

morning on 6/21/07 prior to collection of the 24 hr blo od collection in order to allow placement of Group D

Animals in the hood following dosing of the animals.

Reason for Deviation

Sample analysis results indicate that Group C animals are no longer exhaling ETBE in amounts (above

the assay limit of quantitat ion) which may cross

- contaminate other study samples or expose study

personnel to ETBE. This also affords dosing of Group D animals earlier in the morning.

Deviation

The protocol states (section 10.0) that:

“The statistical analysis to be used will be a dded by amendment” The following staticial analysis will be

performed. Statistical comparison of the parameters Tmax, Cmax, t1/2 Lambda z, Lambda z, AUCall,

AUCobserved and AUCobserved/Dose will be conducted using the Student’s t

- test available in

SAS® Version 9 (SAS Institute Inc., 2004, 2005, 2006a,b). The males will be compared to the

females within dose group for each parameter. Additionally, for the parameter AUCobserved/Dose the

low dose will be compared to the high dose within each sex.

SAS Inst itute Inc. (2004). SAS/STAT® User’s Guide, Version 9.1, Cary, NC: 5136 pp.

SAS Institute Inc. (2005). SAS® Language Reference: Concepts, Version 9.1.3, Cary, NC: 664 pp.

SAS Institute Inc. (2006a). SAS® Language Reference: Dictionary, Version 9.1.3, Cary, NC: 1908 pp.

SAS Institute Inc. (2006b). SAS® Procedures Guide, Version 9.1.3, Cary, NC: 1934 pp.

Reason for Deviation

To expedite statistical analysis, the proposed statistical analysis was agreed with the sponsor.

Deviation

At the following time points for the respective animals, blood was collected from the tail vein instead of the

cannula.

8h (AM01, BF03, DF05),

24h (AM01, AM02, AM03, AM04, BF03, BF04, DF02)RTI Project No.: 0209 4 08.00 7 Protocol No. RTI

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- 23 15m (DF05), 30m (DF05), 4h(BF03)

Reason for Deviation

Cannulae failed in all cases. For the earlier timepoints, a sufficient number of extra animals were not

available to complete the study by dosing additional animals. For 8

- 24h timepoints, tail vein bleeds were

considered an appropriate method of blood collection in the absence of a patent cannula, and a complete

time course to 24h was desired.

Deviation

Periodic stability analyses of the ETBE test substance were conducted via Gas Chromatography with

Flame Ionization Detection (GC

- FID) instead of GC

- MS, and Proton

- NMR was not used.

Reas on for Deviation

The GC

- MS was in constant use during method validations and study sample analyses during this study.

GC

- FID was considered an appropriate alternative and provided a quantitative method for assessing

purity.

Deviation

Group A animals w ere released from quarantine on the same day of receipt.

Reason for Deviation

Animals were not available from the vendor on a prior day to allow for one day between receipt and and

release from quarantine

. The animals were administered ETBE the day foll owing release.

Deviation

Dose formulations were stored in a refrigerator at ca. five degrees Celcius overnight between preparation

and analysis. The protocol stated room temperature.

Reason for Deviation

The test substance is very volatile, and a lower temperature was considered prudent to provide additional

assurance that ETBE remained in the formulation between preparation and dosing. Both room

temperature and refrigerator temperature had been evaluated and both were acceptable.RTI Project No.: 0209 4 08.00 7 Protocol No. RTI

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- 1 Appendix C

Method Validation Plan

Analytical Method

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- 31RTI Project No.: 0209 4 08.00 7 Protocol No. RTI

- 93 2 Final Report

C

- 32RTI Project No.: 0209 4 08.00 7 Protocol No. RTI

- 93 2 Final Report

C

- 33RTI Project No.: 0209 4 08.00 7 Protocol No. RTI

- 93 2 Final Report

C

- 34RTI Project No.: 0209 4 08.00 7 Protocol No. RTI

- 93 2 Final Report

C

- 35RTI Project No.: 0209 4 08.00 7 Protocol No. RTI

- 93 2 Final Report

C

- 36RTI Project No.: 0209 4 08.00 7 Protocol No. RTI

- 93 2 Final Report

C

- 37RTI Project No.: 0209 4 08.00 7 Protocol No. RTI

- 93 2 Final Report

C

- 38RTI Project No.: 0209 4 08.00 7 Protocol No. RTI

- 93 2 Final Report

C

- 39RTI Project No.: 0209 4 08.00 7 Protocol No. RTI

- 93 2 Final Report

C

- 40RTI Project No.: 0209 4 08.00 7 Protocol No. RTI

- 93 2 Final Report

C

- 41RTI Project No.: 0209 4 08.00 7 Protocol No. RTI

- 93 2 Final Report

C

- 42RTI Project No.: 0209 4 08.00 7 Protocol No. RTI

- 93 2 Final Report

C

- 43RTI Project No.: 0209 4 08.00 7 Protocol No. RTI

- 93 2 Final Report

C

- 4 4RTI Project No.: 0209 4 08.00 7 Protocol No. RTI

- 93 2 Final Report

C

- 45RTI Project No.: 0209 4 08.00 7 Protocol No. RTI

- 93 2 Final Report

C

- 46RTI Project No.: 0209 4 08.00 7 Protocol No. RTI

- 93 2 Final Report

D

- 1 Appendix D

Pharmacokinetic AnalysisRTI Project No.: 0209 4 08.00 7 Protocol No. RTI

- 93 2 Final Report

D

- 2 Input File: Data

- [C: \ WINNONLN \ USERCOMP \ ETBE \ ETBEDATA.WDO]

Animal=AM01

Start Time: 12:33:41 07

- 15

- 2007

End Time: 12:33:42 07

- 15

- 2007

WINNONLIN NONCOMPARTMENTAL ANALYSIS PROGRAM (V01.5A)

Core Version 29Oct97

Listing of input commands

TITLE 1

0209408.007, ETBE, RTI

- 932, Rats AM01

- DF05 Analysis 1

MODEL 200

DATE

TIME

NVARIABLES 5

NPOINTS 100

XNUMBER 2

YNUMBER 3

DTIME 0

NCONSTANTS 1

CONSTANTS 49.518

METHOD 2 'Linear trapezoidal

MISSING 'Missing'

NOBSERVATIONS 5

DATA 'WINNLIN.DAT'

BEGINRTI Project No.: 0209 4 08.00 7 Protocol No. RTI

- 93 2 Final Report

D

- 3 Date: 07/15/2007

Time: 12:33:41

0209408.007, ETBE, RTI

- 932, Rats AM01

- DF05 Analysis 1

WINNONLIN NONCOMPARTMENTAL ANALYSIS PROGRAM

Noncompartmental Analysis for Extravascular Administration

Linear Trapezoidal Rule Used to Compute AUC, AUMC

X Y Pred. Res. AUC AUMC WEIG HT

----------------------------------------------------------------------------

.0000 .0000 .0000 .0000

.2600 * 5.914 3.887 2.028 .7689 .1999 1.000

.5122 * 3.588 3.217 .3711 1.967 .6256 1.000

.9850 * 1.732 2.257

- .5248 3.225 1.464 1.000

2.053 * .5660 1.014

- .4482 4.452 2.994 1.000

4.054 * .3114 .2264 .8503E

- 01 5.330 5.420 1.000

----------------------------------------------------------------------------

*) Starred values were included in estimation of Lambda_z.

@) Note

- the concentration at time zero (DTIME) was added for extrapolation

purposes

Dosing_ time .0000

Rsq .8849

Rsq(adjusted) .8465

Corr(x:y)

- .9407

Tlag .0000

Tmax .2600

Cmax 5.9143

No._points_Lambda_z 5

Tlast 4.0536

Clast .3114

AUClast 5.3297

Lambda_z .7495

Lambda_ z_lower .2600

Lambda_z_upper 4.0536

t1/2_Lambda_z .9249

AUCall 5.3297

AUCINF(observed) 5.7452

AUCINF(observed)/D .1160

AUC_%Extrap (obs.) 7.2320

Vz(observed)/F 11.5003

Cl(observed)/F 8.6190

AUCINF(predicted) 5.6318

AUCINF(predicted)/D .1137

AUC_%Extrap(pred.) 5.3631

Vz(predicted)/F 11.7319

Cl(predicted)/F 8.7926

AUMClast 5.4198

AUMCINF(observed) 7.6584

AUMC_%Extrap(obs.) 29.2310

AUMCINF(predicted) 7.0471

AUMC_%Extrap(pred.) 23.0923

MRTlast 1.0169

MRTINF(observed) 1.3330

MRTINF(predicted) 1.2513RTI Project No.: 0209 4 08.00 7 Protocol No. RTI

- 93 2 Final Report

D

- 4 NORMAL ENDINGRTI Project No.: 0209 4 08.00 7 Protocol No. RTI

- 93 2 Final Report

D

- 5 Input File: Data

- [C: \ WINNONLN \ USERCOMP \ ETBE \ ETBEDATA.WDO]

Animal=A M02

Start Time: 12:33:42 07

- 15

- 2007

End Time: 12:33:43 07

- 15

- 2007

WINNONLIN NONCOMPARTMENTAL ANALYSIS PROGRAM (V01.5A)

Core Version 29Oct97

Listing of input commands

TITLE 1

0209408.007, ETBE, RTI

- 932, Rats AM01

- DF05 Analysis 1

MODEL 200

DATE

TIME

NVARIABLES 5

NPOINTS 100

XNUMBER 2

YNUMBER 3

DTIME 0

NCONSTANTS 1

CONSTANTS 50.958

METHOD 2 'Linear trapezoidal

MISSING 'Missing'

NOBSERVATIONS 5

DATA 'WINNLIN.DAT'

BEGINRTI Project No.: 0209 4 08.00 7 Protocol No. RTI

- 93 2 Final Report

D

- 6 Date: 07/15/2007

Time: 12:33:42

0209408.007, ETBE, RTI

- 932, Rats AM01

- DF05 Analysis 1

WINNONLIN NONCOMPARTMENTAL ANALYSIS PROGRAM

Noncompartmental Analysis for Extravascular Administration

Linear Trapezoidal Rule Used to Compute AUC, AUMC

X Y Pred. Res. AUC AUMC WEIGHT

----------------------------------------------------------------------------

.0000 .0000 .0000 .0000

.2386 5.949 .7098 .1694

.4903 * 3.038 2.540 .4976 1.841 .5354 1.000

.9828 * 1.876 1.810 .6587E

- 01 3.051 1.356 1.000

1.984 * .6302 .9094

- .2792 4.305 2.904 1.000

3.974 * .2692 .2312 .3799E

- 01 5.200 5.214 1.000

----------------------------------------------------------------------------

*) Starred values were included in estimation of Lambda_z.

@) Note

- the concentration at time ze ro (DTIME) was added for extrapolation

purposes

Dosing_time .0000

Rsq .9465

Rsq(adjusted) .9197

Corr(x:y)

- .9729

Tlag .00 00

Tmax .2386

Cmax 5.9491

No._points_Lambda_z 4

Tlast 3.9744

Clast .2692

AUClast 5.2001

Lamb da_z .6879

Lambda_z_lower .4903

Lambda_z_upper 3.9744

t1/2_Lambda_z 1.0076

AUCall 5.2001

AUCINF(observed) 5.5914

AUCINF(observed)/D .1097

AUC_%Extrap(obs.) 6.9985

Vz(observed)/F 13.2485

Cl(observed)/F 9.1136

AUCINF(predicted) 5.5362

AUCINF(predicted)/D .1086

AUC _%Extrap(pred.) 6.0707

Vz(predicted)/F 13.3807

Cl(predicted)/F 9.2046

AUMClast 5.2136

AUMCINF(observed) 7.3377

AUMC_%Extrap(obs.) 28.9478

AUMCINF (predicted) 7.0379

AUMC_%Extrap(pred.) 25.9212

MRTlast 1.0026

MRTINF(observed) 1.3123

MRTINF(predicted) 1.2713RTI Project No.: 0209 4 08.00 7 Protocol No. RTI

- 93 2 Final Report

D

- 7 NORMAL ENDINGRTI Project No.: 0209 4 08.00 7 Protocol No. RTI

- 93 2 Final Report

D

- 8 Input File: Data

- [C: \ WINNONLN \ US ERCOMP \ ETBE \ ETBEDATA.WDO]

Animal=AM03

Start Time: 12:33:43 07

- 15

- 2007

End Time: 12:33:44 07

- 15

- 2007

WINNONLIN NONCOMPARTMENTAL ANALYSIS PROGRAM (V01.5A)

Core Version 29Oct97

Listing of input commands

TITLE 1

0209408.007, ETBE, RTI

- 932, Rats AM01

- DF05 Analysis 1

MODEL 200

DATE

TIME

NVARIABLES 5

NPOINTS 100

XNUMBER 2

YNUMBER 3

DTIME 0

NCONSTANTS 1

CONSTANTS 50.052

METHOD 2 'Linear trapezoidal

MISSING 'Missing'

NOBSERVATIONS 5

DATA 'WINNLIN.DAT'

BEGINRTI Project No.: 0209 4 08.00 7 Protocol No. RTI

- 93 2 Final Report

D

- 9 Date: 07/15/2007

Time: 12:33:43

0209408.007, ETBE, RTI

- 932, Rats AM01

- DF05 Analysis 1

WINNONLIN NONCOMPARTMENTAL ANALYSIS PROGRAM

Noncompartmental Analysis for Extravascular Administration

Linear Trapezoidal Rule Used to Compute AUC, AUMC

X Y Pred. Res. AUC AUMC WEIGHT

-------------------------------------------- --------------------------------

.0000 .0000 .0000 .0000

.2358 * 7.435 5.513 1.922 .8767 .2068 1.000

.4950 * 4.991 4.380 .6103 2.487 .7541 1.000

. 9939 * 2.358 2.814

- .4557 4.320 1.955 1.000

1.986 * .6902 1.167

- .4764 5.833 3.798 1.000

3.983 * .2605 .1985 .6204E

- 01 6.782 6.203 1.000

--------------------------- -------------------------------------------------

*) Starred values were included in estimation of Lambda_z.

@) Note

- the concentration at time zero (DTIME) was added for extrapolation

purposes

Dosing_time .0000

Rsq .9373

Rsq(adjusted) .9164

Corr(x:y)

- .9682

Tlag .0000

Tmax .2358

Cmax 7.4352

No._points_Lambda_z 5

Tlast 3.9828

Clast .2605

AUClast 6.7819

Lambda_z .8871

Lambda_z_lower .2358

Lambda_ z_upper 3.9828

t1/2_Lambda_z .7813

AUCall 6.7819

AUCINF(observed) 7.0756

AUCINF(observed)/D .1414

AUC_%Extrap(obs.) 4.1508

Vz(observed )/F 7.9740

Cl(observed)/F 7.0739

AUCINF(predicted) 7.0056

AUCINF(predicted)/D .1400

AUC_%Extrap(pred.) 3.1940

Vz(predicted)/F 8.0536

Cl(predicted)/F 7.1445

AUMClast 6.2028

AUMCINF(observed) 7.7035

AUMC_%Extrap(obs.) 19.4816

AUMCINF(predicted) 7.3462

AUMC_%Extrap(pred.) 15.5650

MRT last .9146

MRTINF(observed) 1.0888

MRTINF(predicted) 1.0486RTI Project No.: 0209 4 08.00 7 Protocol No. RTI

- 93 2 Final Report

D

- 10 NORMAL ENDINGRTI Project No.: 0209 4 08.00 7 Protocol No. RTI

- 93 2 Final Report

D

- 11 Input File: Data

- [C: \ WINNONLN \ USERCOMP \ ETBE \ ETBEDATA.WDO]

Animal=AM04

Start Time: 12:33:44 07

- 15

- 2007

End Time: 12:33:45 07

- 15

- 2007

WINNONLIN NONCOMPARTMENTAL ANALYSIS PROGRAM (V01.5A)

Core Version 29Oct97

Listing of input commands

TITLE 1

0209408.007, ETBE, RTI

- 932, Rats AM01

- DF05 Analysis 1

MODEL 200

DATE

TIME

NVARIABLES 5

NPOINTS 100

XNUMBER 2

YNUMBER 3

DTIME 0

NCONSTANTS 1

CONSTANTS 48.127

METHOD 2 'Linear trapezoidal

MISSING 'Missing'

NOBSERVATIONS 5

DATA 'WINNLIN.DAT'

BEGINRTI Project No.: 0209 4 08.00 7 Protocol No. RTI

- 93 2 Final Report

D

- 12 Date: 07/15/2007

Time: 12:33:44

0209408.007, ETBE, RTI

- 932, Rats AM01

- DF05 Analysis 1

WINNONLIN NONCOMPARTMENTAL ANALYSIS PROGRAM

Noncompartmental Analysis for Extravascular Administration

Linear Trapezoidal Rule Used to Compute AUC, AUMC

X Y Pred. Res. AUC AUMC WEIGHT

----------------------------------------------------------------------------

.0000 .0000 .0000 .0000

.2364 6.758 .7987 .1888

.5108 2.970 2.134 .6162

1.018 * 1.381 1.234 .1463 3.236 1.357 1.000

1.990 * .6 732 .7947

- .1215 4.235 2.691 1.000

4.009 * .3361 .3185 .1765E

- 01 5.253 5.403 1.000

----------------------------------------------------------------------------

*) Starred values were included in estim ation of Lambda_z.

@) Note

- the concentration at time zero (DTIME) was added for extrapolation

purposes

Dosing_time .0000

Rsq .9569

Rsq(adjusted) .9139

Corr(x:y)

- .9782

Tlag .0000

Tmax .2364

Cmax 6.7576

No._points_Lambda_z 3

Tlast 4.0086

Clast .3361

AUClast 5.2534

Lambda_z .4530

Lambda_z_lower 1.0178

Lambda_z_upper 4.0086

t1/2_Lambda_z 1.5302

AUC all 5.2534

AUCINF(observed) 5.9954

AUCINF(observed)/D .1246

AUC_%Extrap(obs.) 12.3763

Vz(observed)/F 17.7209

Cl(observed)/F 8.0274

AUCINF(predicted) 5.9564

AUCINF(predicted)/D .1238

AUC_%Extrap(pred.) 11.8031

Vz(predicted)/F 17.8368

Cl(predicted)/F 8.0799

AUMClast 5.4032

AUM CINF(observed) 10.0156

AUMC_%Extrap(obs.) 46.0523

AUMCINF(predicted) 9.7734

AUMC_%Extrap(pred.) 44.7154

MRTlast 1.0285

MRTINF(observed) 1.6706

MRTINF( predicted) 1.6408RTI Project No.: 0209 4 08.00 7 Protocol No. RTI

- 93 2 Final Report

D

- 13 NORMAL ENDINGRTI Project No.: 0209 4 08.00 7 Protocol No. RTI

- 93 2 Final Report

D

- 14 Input File: Data

- [C: \ WINNONLN \ USERCOMP \ ETBE \ ETBEDATA.WDO]

Animal=BF01

Start Time: 12:33:45 07

- 15

- 2007

End Time: 12:33:46 07

- 15

- 2007

WINNONLIN NONCOMPARTMENTAL ANALYSIS PROGRAM (V01.5A)

Core Version 29Oct97

Listing of input commands

TITLE 1

0209408.007, ETBE, RTI

- 932, Rats AM01

- DF05 Analysis 1

MODEL 200

DATE

TIME

NVARIABLES 5

NPOINTS 100

XNUMBER 2

YNUMBER 3

DTIME 0

NCONSTANTS 1

CONSTANTS 50.286

METHOD 2 'Linear trapezoidal

MISSING 'Missing'

NOBSERVATIONS 5

DATA 'WINNLIN.DAT'

BEGINRTI Project No.: 0209 4 08.00 7 Protocol No. RTI

- 93 2 Final Report

D

- 15 Date: 07/15/2007

Time: 12:33:45

0209408.007, ETBE, RTI

- 932, Rats AM01

- DF05 Analysis 1

WINNONLIN NONCOMPARTMENTAL ANALYSIS PROGRAM

Noncompartmental Analysis for Extravascular Administration

Linear Trapezoidal Rule Used to Compute AUC, AUMC

X Y Pred. Res. AUC AUMC WEIGHT

----------------------------------------------------------------------------

.0000 .0000 .0000 .0000

.2300 5.163 .5937 .1366

.4733 4.118 1.723 .5182

.9847 * 1.951 2.096

- .1453 3.275 1.508 1.000

1.979 * 1.098 .9875 .1105 4.790 3.542 1.000

4.064 * .1966 .2035

- .6847E

- 02 6.140 6.641 1.000

----------------------------------------------------------------------------

*) Starred values were included in estimation of Lambda_z.

@) Note

- the concentration at time ze ro (DTIME) was added for extrapolation

purposes

Dosing_time .0000

Rsq .9938

Rsq(adjusted) .9877

Corr(x:y)

- .9969

Tlag .00 00

Tmax .2300

Cmax 5.1629

No._points_Lambda_z 3

Tlast 4.0644

Clast .1966

AUClast 6.1398

Lamb da_z .7573

Lambda_z_lower .9847

Lambda_z_upper 4.0644

t1/2_Lambda_z .9152

AUCall 6.1398

AUCINF(observed) 6.3994

AUCINF(o bserved)/D .1273

AUC_%Extrap(obs.) 4.0566

Vz(observed)/F 10.3757

Cl(observed)/F 7.8580

AUCINF(predicted) 6.4084

AUCINF(predicted)/D .1274

AUC_%Extrap(pred.) 4.1919

Vz(predicted)/F 10.3610

Cl(predicted)/F 7.8469

AUMClast 6.6410

AUMCINF(observed) 8.0389

AUMC_%Extrap(obs.) 17.3890

AUM CINF(predicted) 8.0876

AUMC_%Extrap(pred.) 17.8863

MRTlast 1.0816

MRTINF(observed) 1.2562

MRTINF(predicted) 1.2620RTI Project No.: 0209 4 08.00 7 Protocol No. RTI

- 93 2 Final Report

D

- 16 NORMAL ENDINGRTI Project No.: 0209 4 08.00 7 Protocol No. RTI

- 93 2 Final Report

D

- 17 Input File: Data

- [C: \ WINNONLN \ USERCOMP \ ETBE \ ETBEDATA.WDO]

Animal=BF02

Start Time: 12:33:46 07

- 15

- 2007

End Time: 12:33:47 07

- 15

- 2007

WINNONLIN NONCOMPARTMENTAL ANALYSIS PROGRAM (V01.5A)

Core Version 29Oct97

Listing of input commands

TITLE 1

0209408.007, ETBE, RTI

- 932, Rats AM01

- DF05 Analysis 1

MODEL 200

DATE

TIME

NVARIABLES 5

NPOINTS 100

XNUMBER 2

YNUMBER 3

DTIME 0

NCONSTANTS 1

CONSTANTS 50.466

METHOD 2 'Linear trapezoidal

MISSING 'Missing'

NOBSERVATIONS 5

DATA 'WINNLIN.DAT'

BEGINRTI Project No.: 0209 4 08.00 7 Protocol No. RTI

- 93 2 Final Report

D

- 18 Date: 07/15/2007

Time: 12:33:46

0209408.007, ETBE, RTI

- 932, Rats AM01

- DF05 Analysis 1

WINNONLIN NONCOMPARTMENTAL ANALYSIS PROGRAM

Noncompartmental Analysis for Extravascular Administration

Linear Trapezoidal Rule Used to Compute AUC, AUMC

X Y Pred. Res. AUC AUMC WEIG HT

----------------------------------------------------------------------------

.0000 .0000 .0000 .0000

.2867 4.150 .5948 .1705

.4678 * 2.919 2.846 .7336 E

- 01 1.235 .4019 1.000

.9792 * 2.215 2.053 .1621 2.548 1.306 1.000

1.984 * .9231 1.080

- .1572 4.125 3.316 1.000

4.046 * .3061 .2895 .1660E

- 01 5.392 6.480 1.000

----------------------------------------------------------------------------

*) Starred values were included in estimation of Lambda_z.

@) Note

- the concentration at time zero (DTIME) was added for extrapolation

purposes

Dosing_time .0000

Rsq .9889

Rsq(adjusted) .9834

Corr(x:y)

- .9945

Tlag .0000

Tmax .2867

Cmax 4.1497

No._points_Lambda_z 4

Tlast 4.0456

Clast .3061

AUClast 5.3916

Lambda_z .6388

Lambda_ z_lower .4678

Lambda_z_upper 4.0456

t1/2_Lambda_z 1.0851

AUCall 5.3916

AUCINF(observed) 5.8708

AUCINF(observed)/D .1163

AUC_%Extrap (obs.) 8.1621

Vz(observed)/F 13.4566

Cl(observed)/F 8.5962

AUCINF(predicted) 5.8448

AUCINF(predicted)/D .1158

AUC_%Extrap(pred.) 7.7537

Vz(predicted)/F 13.5164

Cl(predicted)/F 8.6344

AUMClast 6.4801

AUMCINF(observed) 9.1687

AUMC_%Extrap(obs.) 29.3242

AUMCINF(predicted) 9.0229

AUMC_%Extrap(pred.) 28.1819

MRTlast 1.2019

MRTINF(observed) 1.5618

MRTINF(predicted) 1.5438RTI Project No.: 0209 4 08.00 7 Protocol No. RTI

- 93 2 Final Report

D

- 19 NORMAL ENDINGRTI Project No.: 0209 4 08.00 7 Protocol No. RTI

- 93 2 Final Report

D

- 20 Input File: Data

- [C: \ WINNONLN \ USERCOMP \ ETBE \ ETBEDATA.WDO]

Animal=BF03

Start Time: 12:33:47 07

- 15

- 2007

End Time: 12:33:48 07

- 15

- 2007

WINNONLIN NONCOMPARTMENTAL ANALYSIS PROGRAM (V01.5A)

Core Version 29Oct97

Listing of input commands

TITLE 1

0209408.007, ETBE, RTI

- 932, Rats AM01

- DF05 Analysis 1

M ODEL 200

DATE

TIME

NVARIABLES 5

NPOINTS 100

XNUMBER 2

YNUMBER 3

DTIME 0

NCONSTANTS 1

CONSTANTS 51.466

METHOD 2 'Linear trapezoidal

MISSING 'Missing'

NOBSERVATIONS 5

DATA 'WINNLIN.DAT'

BEGINRTI Project No.: 0209 4 08.00 7 Protocol No. RTI

- 93 2 Final Report

D

- 21 Date: 07/15/2007

Time: 12:33:47

0209408.007, ETBE, RTI

- 932, Rats AM01

- DF05 Analysis 1

WINNONLIN NONCOMPARTMENTAL ANALYSIS PROGRAM

Noncompartmental Analysis for Extravascular Administration

Linear Trapezoidal Rule Used to Compute AUC, AUMC

X Y Pred. Res. AUC AUMC WEIGHT

-------------------------------------------- --------------------------------

.0000 .0000 .0000 .0000

.2011 6.164 .6198 .1247

.4869 3.333 1.977 .5338

1.014 * 1.502 1.317 .1846 3.252 1.363 1.000

2.032 * .6970 .8449

- .1479 4.371 2.859 1.000

4.211 * .3473 .3266 .2064E

- 01 5.509 5.996 1.000

------------------------ ----------------------------------------------------

*) Starred values were included in estimation of Lambda_z.

@) Note

- the concentration at time zero (DTIME) was added for extrapolation

purposes

Dosing_time .0000

Rsq .9460

Rsq(adjusted) .8919

Corr(x:y)

- .9726

Tlag .0000

Tmax .2011

Cmax 6.1641

No._points_Lam bda_z 3

Tlast 4.2111

Clast .3473

AUClast 5.5086

Lambda_z .4362

Lambda_z_lower 1.0142

Lambda_z_upper 4.2111

t1/2_Lambda_z 1.5892

AUCall 5.5086

AUCINF(observed) 6.3048

AUCINF(observed)/D .1225

AUC_%Extrap(obs.) 12.6278

Vz(observed)/F 18.7150

Cl(observed)/F 8.1630

AUCINF(predicted) 6.2575

AUCINF(predicted)/D .1216

AUC_%Extrap(pred.) 11.9670

Vz(predicted)/F 18.8566

Cl( predicted)/F 8.2247

AUMClast 5.9957

AUMCINF(observed) 11.1737

AUMC_%Extrap(obs.) 46.3412

AUMCINF(predicted) 10.8659

AUMC_%Extrap(pred.) 44.8213

MRTlast 1.0884

MRTINF(observed) 1.7723

MRTINF(predicted) 1.7365RTI Project No.: 0209 4 08.00 7 Protocol No. RTI

- 93 2 Final Report

D

- 22 NORMAL ENDINGRTI Project No.: 0209 4 08.00 7 Protocol No. RTI

- 93 2 Final Report

D

- 23 Input File: Data

- [C: \ WINNONLN \ USERCOMP \ ETBE \ ETBEDATA.WDO]

Animal=BF04

Start Time: 12:33:48 07

- 15

- 2007

End Time: 12:33:49 07

- 15

- 2007

WINNONLIN NONCOMPARTMENTAL ANALYSIS PROGRAM (V01.5A)

Core Version 29Oct97

Listing of input commands

TITLE 1

0209408.007, ETBE, RTI

- 932, Rats AM01

- DF05 Analysis 1

MODEL 200

DATE

TIME

NVARIABLES 5

NPOINTS 100

XNUMBER 2

YNUMBER 3

DTIME 0

NCONSTANTS 1

CONSTANTS 51.974

METHOD 2 'Linear trapezoidal

MISSING 'Missing'

NOBSERVATIONS 5

DATA 'WINNLIN.DAT'

BEGINRTI Project No.: 0209 4 08.00 7 Protocol No. RTI

- 93 2 Final Report

D

- 24 Date: 07/15/2007

Time: 12:33:48

0209408.007, ETBE, RTI

- 932, Rats AM01

- DF05 Analysis 1

WINNONLIN NONCOMPARTMENTAL ANALYSIS PROGRAM

Noncompartmental Analysis for Extravascular Administration

Linear Trapezoidal Rule Used to Compute AUC, AUMC

X Y Pred. Res. AUC AUMC WEIGHT

----------------------------------------------------------------------------

.0000 .0000 .0000 .0000

.2217 7.872 .8725 .1934

.4969 * 4.048 3.147 .9014 2.513 .7104 1.000

.9892 * 1.984 2.085

- .1012 3.998 1.688 1.000

1.992 * .6272 .9016

- .2744 5.307 3.299 1.000

4.025 * .1935 .1647 .2872E

- 01 6.141 5.360 1.000

----------------------------------------------------------------------------

*) Starred values were included in estimation of Lambda_z.

@) Note

- the concentration at time zero (DTIME) was added for extrapolation

purposes

Dosing_time .0000

Rsq .9582

Rsq(adjusted) .9372

Corr(x:y)

- .9789

Tlag .0000

Tmax .2217

Cmax 7.8718

No._points_Lambda_z 4

Tlast 4.0250

Clast .1935

AUClast 6.1410

Lambda_z .8361

Lambda_z_lower .4969

Lambda_z_upper 4.0250

t1/2_Lambda_z .8290

AUCall 6.1410

AUCINF(observed) 6.3724

AUCINF(observed)/D .1226

AUC_%Extrap(obs.) 3.6311

Vz(observed)/F 9.7553

Cl(observed)/F 8.1562

AUCINF(predicte d) 6.3380

AUCINF(predicted)/D .1219

AUC_%Extrap(pred.) 3.1088

Vz(predicted)/F 9.8082

Cl(predicted)/F 8.2004

AUMClast 5.3604

AUMCINF(observed) 6.5684

AUMC_%Extrap(obs.) 18.3921

AUMCINF(predicted) 6.3891

AUMC_%Extrap(pred.) 16.1014

MRTlast .8729

MRTINF(observed) 1.0308

MRT INF(predicted) 1.0081RTI Project No.: 0209 4 08.00 7 Protocol No. RTI

- 93 2 Final Report

D

- 25 NORMAL ENDINGRTI Project No.: 0209 4 08.00 7 Protocol No. RTI

- 93 2 Final Report

D

- 26 Input File: Data

- [C: \ WINNONLN \ USERCOMP \ ETBE \ ETBEDATA.WDO]

Animal=CM01

Start Time: 12:33:49 07

- 15

- 2007

End Time: 12:33:50 07

- 15

- 2007

WINNONLIN NONCOMPARTMENTAL ANALYSIS PROGRAM (V01. 5A)

Core Version 29Oct97

Listing of input commands

TITLE 1

0209408.007, ETBE, RTI

- 932, Rats AM01

- DF05 Analysis 1

MODEL 200

DATE

TIME

NVARIABLES 5

NPOINTS 100

XNUMBER 2

YNUMBER 3

DTIME 0

NCONSTANTS 1

CONSTANTS 10.311

METHOD 2 'Linear trapezoidal

MISSING 'Missing'

NOBSERVATIONS 4

DATA 'WINNLIN.DAT'

BEGINRTI Project No.: 0209 4 08.00 7 Protocol No. RTI

- 93 2 Final Report

D

- 27 Date: 07/15/2007

Time: 12:33:49

0209408.007, ETBE, RTI

- 932, Rats AM01

- DF05 Analysis 1

WINNONLIN NONCOMPARTMENTAL ANALYSIS PROGRAM

Noncompartmental Analysis for Extravascular Administration

Linear Trapezoidal Rule Used to Compute AUC, AUMC

X Y Pred. Res. AUC AUMC WEIGHT

----------------------------------------------------------------------------

.0000 .0000 .0000 .0000

.2367 1.019 .1206 .2853E

- 01

.4958 * .4226 .3939 .2869E

- 01 .3074 .8693E

- 01 1.000

.9917 * .2188 .2431

- .2432E

- 01 .4664 .1927 1.000

1.985 * .9580E

- 01 .9250E

- 01 .3304E

- 02 .6226 .3949 1.000

----------------------------------------------------------------------------

*) Starred values were included in estimation of Lambda_z.

@) Note

- the concentration at time ze ro (DTIME) was added for extrapolation

purposes

Dosing_time .0000

Rsq .9844

Rsq(adjusted) .9687

Corr(x:y)

- .9922

Tlag .00 00

Tmax .2367

Cmax 1.0189

No._points_Lambda_z 3

Tlast 1.9850

Clast .0958

AUClast .6226

Lambda_z .9730

Lambda_z_lower .4958

Lambda_z_upper 1.9850

t1/2_Lambda_z .7124

AUCall .6226

AUCINF(observed) .7211

AUC INF(observed)/D .0699

AUC_%Extrap(obs.) 13.6549

Vz(observed)/F 14.6965

Cl(observed)/F 14.2990

AUCINF(predicted) .7177

AUCINF(predicted)/D .0696

AUC_%Ex trap(pred.) 13.2464

Vz(predicted)/F 14.7661

Cl(predicted)/F 14.3667

AUMClast .3949

AUMCINF(observed) .6916

AUMC_%Extrap(obs.) 42.8969

AUMCINF(pre dicted) .6813

AUMC_%Extrap(pred.) 42.0394

MRTlast .6342

MRTINF(observed) .9590

MRTINF(predicted) .9493RTI Project No.: 0209 4 08.00 7 Protocol No. RTI

- 93 2 Final Report

D

- 28 NORMAL ENDINGRTI Project No.: 0209 4 08.00 7 Protocol No. RTI

- 93 2 Final Report

D

- 29 Input File: Data

- [C: \ WINNONLN \ USERCOMP \ ETBE \ ETBEDATA.WDO]

Animal=CM02

Start Time: 12:33:50 07

- 15

- 2007

End Time: 12:33:51 07

- 15

- 2007

WINNONLIN NONCOMPARTMENTAL ANALYSIS PROGRAM (V01.5A)

Core Version 29Oct97

Listing of input commands

TITLE 1

0209408.007, ETBE, RTI

- 932, Rats AM01

- DF05 Analysis 1

MODEL 200

DATE

TIME

NVARIABLES 5

NPOINTS 100

XNUMBER 2

YNUMBER 3

DTIME 0

NCONSTANTS 1

CONSTANTS 10.525

METHOD 2 'Linear trapezoidal

MISSING 'Missing'

NOBSERVATIONS 3

DATA 'WINNLIN.DAT'

BEGINRTI Project No.: 0209 4 08.00 7 Protocol No. RTI

- 93 2 Final Report

D

- 30 Date: 07/15/2007

Time: 12:33:50

0209408.007, ETBE, RTI

- 932, Rats AM01

- DF05 Analysis 1

WINNONLIN NONCOMPAR TMENTAL ANALYSIS PROGRAM

Noncompartmental Analysis for Extravascular Administration

Linear Trapezoidal Rule Used to Compute AUC, AUMC

X Y Pred. Res. AUC AUMC WEIGHT

---------------- ------------------------------------------------------------

.0000 .0000 .0000 .0000

.2383 * 1.239 1.077 .1612 .1476 .3518E

- 01 1.000

.4906 * .5035 .6206

- .1171 .36 73 .1036 1.000

.9953 * .2206 .2058 .1485E

- 01 .5500 .2213 1.000

----------------------------------------------------------------------------

*) Starred values were included in estimation of Lambda_z.

@) Note

- the concentration at time zero (DTIME) was added for extrapolation

purposes

Dosing_time .0000

Rsq .9543

Rsq(adjusted) .9087

Corr(x:y)

- .9769

Tlag .0000

Tmax .2383

Cmax 1.2386

No._points_Lambda_z 3

Tlast .9953

Clast .2206

AUClast .5500

Lambda_z 2.1872

Lambda_z_lower .2383

Lambda_z_upper .9953

t1/2_Lambda_z .3169

AUCall .5500

AUCINF(observed) .6509

AUCINF(observed)/D .0618

AUC_%Extrap(obs.) 15.4957

Vz(observed)/F 7.3931

Cl(observed)/F 16.1705

AUCINF(predicted) .6441

AUC INF(predicted)/D .0612

AUC_%Extrap(pred.) 14.6051

Vz(predicted)/F 7.4710

Cl(predicted)/F 16.3409

AUMClast .2213

AUMCINF(observed) .3678

AUMC_%Extrap(obs.) 39.8316

AUMCINF(predicted) .3579

AUMC_%Extrap(pred.) 38.1742

MRTlast .4023

MRTINF(observed) .5651

MRTINF(predicted) .5557RTI Project No.: 0209 4 08.00 7 Protocol No. RTI

- 93 2 Final Report

D

- 31 N ORMAL ENDINGRTI Project No.: 0209 4 08.00 7 Protocol No. RTI

- 93 2 Final Report

D

- 32 Input File: Data

- [C: \ WINNONLN \ USERCOMP \ ETBE \ ETBEDATA.WDO]

Animal=CM03

Start Time: 12:33:51 07

- 15

- 2007

End Time: 12:33:52 07

- 15

- 2007

WINNONLIN NONCOMPARTMENTAL ANALYSIS PROGRAM (V01.5A)

Core Version 29Oct97

Listing of input commands

TITLE 1

0209408.007, ETBE, RTI

- 932, Rats AM01

- DF05 Analysis 1

MODEL 200

DATE

TIME

NVARIABLES 5

NPOINTS 100

XNUMBER 2

YNUMBER 3

DTIME 0

NCONSTANTS 1

CONSTANTS 10.067

METHOD 2 'Linear trapezoidal

MISSING 'Missing'

NOBSERVATIONS 3

DATA 'WINNLIN.DAT'

BEGINRTI Project No.: 0209 4 08.00 7 Protocol No. RTI

- 93 2 Final Report

D

- 33 Date: 07/15/2007

Time: 12:33:51

0209408.007, ETBE, RTI

- 932, Rats AM01

- DF05 Analysis 1

WINN ONLIN NONCOMPARTMENTAL ANALYSIS PROGRAM

Noncompartmental Analysis for Extravascular Administration

Linear Trapezoidal Rule Used to Compute AUC, AUMC

X Y Pred. Res. AUC AUMC WEIGHT

----------------------------------------------------------------------------

.0000 .0000 .0000 .0000

.2381 * 1.135 1.022 .1129 .1351 .3217E

- 01 1.000

.4931 * .5471 .6408

- .9375E

- 01 .3496 .1010 1.000

.9933 * .2704 .2563 .1406E

- 01 .5541 .2357 1.000

----------------------------------------------------------------------------

*) Starred values were included in estimation of Lambda_z

.

@) Note

- the concentration at time zero (DTIME) was added for extrapolation

purposes

Dosing_time .0000

Rsq .9623

Rsq(adjusted) .9245

Corr(x:y)

- .98 10

Tlag .0000

Tmax .2381

Cmax 1.1353

No._points_Lambda_z 3

Tlast .9933

Clast .2704

AUClast .5541

Lambda_z 1.8318

Lambda_z_lower .2381

Lambda_z_upper .9933

t1/2_Lambda_z .3784

AUCall .5541

AUC INF(observed) .7017

AUCINF(observed)/D .0697

AUC_%Extrap(obs.) 21.0340

Vz(observed)/F 7.8318

Cl(observed)/F 14.3462

AUCINF(predicted) .6940

AUCINF( predicted)/D .0689

AUC_%Extrap(pred.) 20.1608

Vz(predicted)/F 7.9185

Cl(predicted)/F 14.5049

AUMClast .2357

AUMCINF(observed) .4629

AUMC_%Extrap(obs.) 49.0838

AUMCINF(predicted) .4511

AUMC_%Extrap(pred.) 47.7502

MRTlast .4253

MRTINF(observed) .6596

MRTINF(predicted) .6499RTI Project No.: 0209 4 08.00 7 Protocol No. RTI

- 93 2 Final Report

D

- 34 N ORMAL ENDINGRTI Project No.: 0209 4 08.00 7 Protocol No. RTI

- 93 2 Final Report

D

- 35 Input File: Data

- [C: \ WINNONLN \ USERCOMP \ ETBE \ ETBEDATA.WDO]

Animal=CM08

Start Time: 12:33:52 07

- 15

- 2007

End Time: 12:33:53 07

- 15

- 2007

WINNONLIN NONCOMPARTMENTAL ANALYSIS PROGRAM (V01.5A)

Core Version 29Oct97

Listing of input commands

TITLE 1

0209408.007, ETBE, RTI

- 932, Rats AM01

- DF05 Analysis 1

MODEL 200

DATE

TIME

NVARIABLES 5

NPOINTS 100

XNUMBER 2

YNUMBER 3

DTIME 0

NCONSTANTS 1

CONSTANTS 10.083

METHOD 2 'Line ar trapezoidal

MISSING 'Missing'

NOBSERVATIONS 4

DATA 'WINNLIN.DAT'

BEGINRTI Project No.: 0209 4 08.00 7 Protocol No. RTI

- 93 2 Final Report

D

- 36 Date: 07/15/2007

Time: 12:33:52

0209408.007, ETBE, RTI

- 932, Rats AM01

- DF05 Analysis 1

WINNONLIN NONCOMPARTMENTAL ANALYSIS PROGRAM

Noncompartmental Analysis for Extravascular Administration

Linear Trapezoidal Rule Used to Compute AUC, AUMC

X Y Pred. Res. AUC AUMC WEIGHT

----------------------------------------------------------------------------

.0000 .0000 .0000 .0000

.2733 .8336 .1139 .3114E

- 01

.5261 * .4362 .3840 .5216E

- 01 .2744 .8894E

- 01 1.000

.9919 * .1903 .2293

- .3899E

- 01 .4203 .1863 1.000

1.996 * .7999E

- 01 .7540E

- 01 .4589E

- 02 .5560 .3613 1.000

----------------------------------------------------------------------------

*) Starred values were included in estimation of Lambda_z.

@) Note

- the concentration at time zero (DTIME) was added for extrapolation

purposes

Dosing_time .0000

Rsq .9621

Rsq(adjusted) .9243

Corr(x:y)

- .9809

Tlag .0000

Tmax .2733

Cma x .8336

No._points_Lambda_z 3

Tlast 1.9961

Clast .0800

AUClast .5560

Lambda_z 1.1074

Lambda_z_lo wer .5261

Lambda_z_upper 1.9961

t1/2_Lambda_z .6259

AUCall .5560

AUCINF(observed) .6282

AUCINF(observed)/D .0623

AUC_%Extrap(obs .) 11.4983

Vz(observed)/F 14.4933

Cl(observed)/F 16.0494

AUCINF(predicted) .6241

AUCINF(predicted)/D .0619

AUC_%Extrap(pred.) 10.9106

Vz(predicted)/F 14.5896

Cl(predicted)/F 16.1560

AUMClast .3613

AUMCINF(observed) .5707

AUMC_%Extrap(obs.) 36.6964

AUMCINF(predicted) .5587

AUM C_%Extrap(pred.) 35.3351

MRTlast .6498

MRTINF(observed) .9084

MRTINF(predicted) .8952RTI Project No.: 0209 4 08.00 7 Protocol No. RTI

- 93 2 Final Report

D

- 37 NORMAL ENDINGRTI Project No.: 0209 4 08.00 7 Protocol No. RTI

- 93 2 Final Report

D

- 38 Input File: Data

- [C: \ WINNONLN \ USERCOMP \ ETBE \ ETBEDATA.WDO]

Animal=DF01

Start Time: 12:33:53 07

- 15

- 2007

End Time: 12:33:54 07

- 15

- 2007

WINNONLIN NONCOMPARTMENTAL ANALYSIS PROGRAM (V01.5A)

Core Version 29Oct97

Listing of input commands

TITLE 1

0209408.007, ETBE, RTI

- 932, Rats AM01

- DF05 Analysis 1

MODEL 200

DATE

TIME

NVARIABLES 5

NPOINTS 100

XNUMBER 2

YNUMBER 3

DTIME 0

NCONSTANTS 1

CONSTANTS 10.126

METHOD 2 'Linear trapezoidal

MISSING 'Missing'

NOBSERVATIONS 4

DATA 'WINNLIN.DAT'

BEGINRTI Project No.: 0209 4 08.00 7 Protocol No. RTI

- 93 2 Final Report

D

- 39 Date: 07/15/2007

Time: 12:33:53

0209408.007, ETBE, RTI

- 932, Rats AM01

- DF05 Analysis 1

WINNONLIN NONCOMPARTMENTAL ANALYSIS PROGRAM

Noncompartmental Analysis for Extravascular Administration

Linear Trapezoidal Rule Used to Compute AUC, AUMC

X Y Pred. Res. AUC AUMC WEIGHT

-------------------------------------------- --------------------------------

.0000 .0000 .0000 .0000

.2436 1.255 .1529 .3725E

- 01

.5014 * .6526 .6075 .4507E

- 01 .3989 .1188 1.000

.9822 * .3202 .3559

- .3572E

- 01 .6327 .2731 1.000

1.989 * .1202 .1162 .4040E

- 02 .8545 .5519 1.000

----------------------------------------------------------------------------

*) Starred values were included in estimation of Lambda_z.

@) Note

- the concentration at time zero (DTIME) was added for extrapolation

purposes

Dosing_time .0000

Rsq .9879

Rsq(adjusted) .9758

Corr(x:y)

- .9939

Tlag .0000

Tmax .2436

Cmax 1.2555

No._points_Lambda_z 3

Tlast 1.9892

Clast .1202

AUClast .8545

Lambda_z 1.1118

Lambda_z_lower .5014

Lambda_z_upper 1.9892

t1/2_Lambda_z .6234

AUC all .8545

AUCINF(observed) .9626

AUCINF(observed)/D .0951

AUC_%Extrap(obs.) 11.2341

Vz(observed)/F 9.4613

Cl(observed)/F 10.5191

AUCINF( predicted) .9590

AUCINF(predicted)/D .0947

AUC_%Extrap(pred.) 10.8978

Vz(predicted)/F 9.4972

Cl(predicted)/F 10.5589

AUMClast .5519

AUMCINF(obs erved) .8643

AUMC_%Extrap(obs.) 36.1437

AUMCINF(predicted) .8538

AUMC_%Extrap(pred.) 35.3587

MRTlast .6459

MRTINF(observed) .8978

MRTINF(predicte d) .8903RTI Project No.: 0209 4 08.00 7 Protocol No. RTI

- 93 2 Final Report

D

- 40 NORMAL ENDINGRTI Project No.: 0209 4 08.00 7 Protocol No. RTI

- 93 2 Final Report

D

- 41 Input File: Data

- [C: \ WINNONLN \ USERCOMP \ ETBE \ ETBEDATA.WDO]

Animal=DF02

Start Time: 12:33:54 07

- 15

- 2007

End Time: 12:33:55 07

- 15

- 2007

WINNONLIN NONCOMPARTMENTAL ANALYSIS PROGRAM (V01.5A)

Core Version 29Oct97

Listing of input commands

TITLE 1

0209408.007, ETBE, RTI

- 932, Rats AM01

- DF05 Analysis 1

MODEL 200

DATE

TIME

NVARIABLES 5

NPOINTS 100

XNUMBER 2

YNUMBER 3

DTIME 0

NCONSTANTS 1

CONSTANTS 10.302

METHOD 2 'Linear trapezoidal

MISSING 'Missing'

NOBSERVATIONS 3

DATA 'WINNLIN.DAT'

BEGINRTI Project No.: 0209 4 08.00 7 Protocol No. RTI

- 93 2 Final Report

D

- 42 Date: 07/15/2007

Time: 12:33:54

0209408.007, ETBE, RTI

- 932, Rats AM01

- DF05 Analysis 1

WINNONLIN NONCOMPARTMENTAL ANALYSIS PROGRAM

Noncompartmental Analysis for Extravascular Administration

Linear Trapezoidal Rule Used to Compute AUC, AUMC

X Y Pred. Res . AUC AUMC WEIGHT

----------------------------------------------------------------------------

.0000 .0000 .0000 .0000

.2442 * 1.046 .9611 .8478E

- 01 .1277 .3118E

- 01 1.000

.4761 * .5544 .6273

- .7292E

- 01 .3133 .9140E

- 01 1.000

.9783 * .2589 .2490 .9914E

- 02 .5175 .2213 1.000

----------------------------------------------------------------------------

*) Starred values were included in estimation of Lambda_z.

@) Note

- the concentration at time zero (DTIME) was added for extrapolation

purposes

Dosing_time .0000

Rsq .9755

Rsq(adjusted) .9510

Corr(x:y)

- .9877

Tlag .0000

Tmax .2442

Cmax 1.0459

No._points_Lambda_z 3

Tlast .9783

Clast .2589

AUClast .5175

Lambda_z 1.8395

Lambda_z_lower .2442

Lambda_z_upper .9783

t1/2_Lambda_z .3768

AUCall .5175

AUCINF(observed) .6583

AUCINF(observed)/D .0639

AUC_%Extrap(obs.) 21.3847

Vz(observed)/F 8.5079

Cl(observed)/F 15.6503

AUCINF(predicted) .6529

AUCINF(predicted)/D .0634

AUC_%Extrap(pred.) 20.7357

Vz(predicted)/F 8.5781

Cl(predicted)/F 15.7795

AUMClast .2213

AUMCINF(observed) .4355

AUMC_%Extrap(obs.) 49.1905

AUMCINF(predicted) .4273

AUMC_%Extrap(pred.) 48.2151

MRTlast .4276

MRT INF(observed) .6616

MRTINF(predicted) .6545RTI Project No.: 0209 4 08.00 7 Protocol No. RTI

- 93 2 Final Report

D

- 43 NORMAL ENDINGRTI Project No.: 0209 4 08.00 7 Protocol No. RTI

- 93 2 Final Report

D

- 44 Input File: Data

- [C: \ WINNONLN \ USERCOMP \ ETBE \ ETBEDATA.WDO]

Animal=DF04

Start Time: 12:33:55 07

- 15

- 2007

End Time: 12:33:56 07

- 15

- 2007

WINNONL IN NONCOMPARTMENTAL ANALYSIS PROGRAM (V01.5A)

Core Version 29Oct97

Listing of input commands

TITLE 1

0209408.007, ETBE, RTI

- 932, Rats AM01

- DF05 Analysis 1

MODEL 200

DATE

TIME

NVARIABLES 5

NPOINTS 100

XNUMBER 2

YNUMBER 3

DTIME 0

NCONSTANTS 1

CONSTANTS 10.122

METHOD 2 'Linear trapezoidal

MISSING 'Missing'

NOBSERVATIONS 4

DATA 'WINNLIN.DAT'

BEGINRTI Project No.: 0209 4 08.00 7 Protocol No. RTI

- 93 2 Final Report

D

- 45 Date: 07/15/2007

T ime: 12:33:55

0209408.007, ETBE, RTI

- 932, Rats AM01

- DF05 Analysis 1

WINNONLIN NONCOMPARTMENTAL ANALYSIS PROGRAM

Noncompartmental Analysis for Extravascular Administration

Linear Trapezoidal Rule Used to Compute AUC, AUMC

X Y Pred. Res. AUC AUMC WEIGHT

----------------------------------------------------------------------------

.0000 .0000 .0000 .0000

.2839 1.008 .1430 .4061E

- 01

.4981 * .4603 .4292 .3110E

- 01 .3002 .9579E

- 01 1.000

.9722 * .2453 .2714

- .2614E

- 01 .4675 .2067 1.000

2.032 * .1006 .9753 E

- 01 .3102E

- 02 .6508 .4413 1.000

----------------------------------------------------------------------------

*) Starred values were included in estimation of Lambda_z.

@) Note

- the concentration at time zero (DTIME) was added for extrapo lation

purposes

Dosing_time .0000

Rsq .9862

Rsq(adjusted) .9724

Corr(x:y)

- .9931

Tlag .0000

Tmax .2839

Cmax 1.0077

No._points_Lambda_z 3

Tlast 2.0317

Clast .1006

AUClast .6508

Lambda_ z .9662

Lambda_z_lower .4981

Lambda_z_upper 2.0317

t1/2_Lambda_z .7174

AUCall .6508

AUCINF(observed) .7549

AUCINF(obse rved)/D .0746

AUC_%Extrap(obs.) 13.7965

Vz(observed)/F 13.8773

Cl(observed)/F 13.4080

AUCINF(predicted) .7517

AUCINF(predicted)/D .0743

AUC_%Extrap(pre d.) 13.4283

Vz(predicted)/F 13.9366

Cl(predicted)/F 13.4653

AUMClast .4413

AUMCINF(observed) .7607

AUMC_%Extrap(obs.) 41.9869

AUMCINF(predicted) .7509

AUMC_%Extrap(pred.) 41.2262

MRTlast .6781

MRTINF(observed) 1.0077

MRTINF(predicted) .9989RTI Project No.: 0209 4 08.00 7 Protocol No. RTI

- 93 2 Final Report

D

- 46 NORMAL ENDINGRTI Project No.: 0209 4 08.00 7 Protocol No. RTI

- 93 2 Final Report

D

- 47 Input File: Data

- [C: \ WINNONLN \ USERCOMP \ ETBE \ ETBEDATA.WDO]

Animal=DF05

Start Time: 12:33:57 07

- 15

- 2007

End Time: 12:33:57 07

- 15

- 2007

WINNONLIN NONCOMPARTMENTAL ANALYSIS PROGRAM (V01.5A)

Core Version 29Oct97

Listing of input commands

TITLE 1

0209408.007, ETBE, RTI

- 932, Rats AM01

- DF05 Analysis 1

MODEL 200

DATE

TIME

NVARIABLES 5

NPOINTS 100

XNUMBER 2

YNUMBER 3

DTIME 0

NCONSTANTS 1

CONSTANTS 10.239

METHOD 2 'Linear trapezoidal

MISSING 'Missing'

NOBSERVATIONS 3

DATA 'WINNLIN.DAT'

BEGINRTI Project No.: 0209 4 08.00 7 Protocol No. RTI

- 93 2 Final Report

D

- 48 Date: 07/15/2007

Time: 12:33:56

0209408.007, ETBE, RTI

- 932, Rats AM01

- DF05 Analysis 1

WINNONLIN NONCOMPAR TMENTAL ANALYSIS PROGRAM

Noncompartmental Analysis for Extravascular Administration

Linear Trapezoidal Rule Used to Compute AUC, AUMC

X Y Pred. Res. AUC AUMC WEIGHT

---------------- ------------------------------------------------------------

.0000 .0000 .0000 .0000

.3975 * .2818 .2952

- .1344E

- 01 .5600E

- 01 .2226E

- 01 1.000

.4908 * .3078 .2910 .1678E

- 01 .8351E

- 01 .3454E

- 01 1.000

.9517 * .2686 .2711

- .2547E

- 02 .2163 .1282 1.000

----------------------------------------------------------------------------

*) Starred values were included in estimation of Lambda_z.

@) Note

- the concentration at time zero (DTIME) was added for extrapolation

purposes

Dosing_time .0000

Rsq .4346

Rsq(adjusted)

- .1309

Cor r(x:y)

- .6592

Tlag .0000

Tmax .4908

Cmax .3078

No._points_Lambda_z 3

Tlast .9517

Clast .2686

AUClast .2163

Lambda_z .1536

Lambda_z_lower .3975

Lambda_z_upper .9517

t1/2_Lambda_z 4.5134

AUC all .2163

AUCINF(observed) 1.9651

AUCINF(observed)/D .1919

AUC_%Extrap(obs.) 88.9924

Vz(observed)/F 33.9275

Cl(observed)/F 5.2104

AUCINF(predicted) 1.9817

AUCINF(predicted)/D .1935

AUC_%Extrap(pred.) 89.0845

Vz(predicted)/F 33.6436

Cl(predicted)/F 5.1668

AUMClast .1282

AUM CINF(observed) 13.1797

AUMC_%Extrap(obs.) 99.0270

AUMCINF(predicted) 13.3035

AUMC_%Extrap(pred.) 99.0361

MRTlast .5928

MRTINF(observed) 6.7069

MRTINF( predicted) 6.7132RTI Project No.: 0209 4 08.00 7 Protocol No. RTI

- 93 2 Final Report

D

- 49 NORMAL ENDINGRTI Project No.: 0209 4 08.00 7 Protocol No. RTI

- 93 2 Final Report

D

- 50RTI Project No.: 0209 4 08.00 7 Protocol No. RTI

- 93 2 Final Report

D

- 51RTI Project No.: 0209 4 08.00 7 Protocol No. RTI

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- 1 Appendix E

Dose Formulation AnalysisRTI Project No.: 0209 4 08.00 7 Protocol No. RTI

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- 2 Dose formulation analysis was conducted by gas chromatography with flame ionization detection as

described in Project Specific Analytical Method for Analysis of Ethyl Tertiary Butyl Ether in Oral Dose

Formulations, AM

- D0209408.007.0

.

Prior to conduct of the in

- life portion of the study, the method was used to determine the stability of mock

dose formulations at both the low dose (1 mg/ml) and the high dose (5 mg/ml). The dose solutions were

found to be stable stored both at room temperature for up to three days and in a refrigerator for 1 day

(Table 1).

The dose formulation analysis results are tabulated for each dose solution in Tables 2

– 5. The

associated calibration curves and data are shown in Figures 1

– 4.

The Project Specific Analytical Method for Analysis of Ethyl Tertiary Butyl Ether in Oral Dose

Formulations, AM

- D0209408.007.0 is shown at the en d of this Appendix.RTI Project No.: 0209 4 08.00 7 Protocol No. RTI

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- 3 Table 1. Dose formulation s tability

.

Mock Dose. Storage condition Concentration of

ETBE (  g/ml) SD Stability (%) a

1.0 mg/mL 0 day 1141 32

-

1.0 mg/mL Ca. 4 o C, 1 day 1156 60 101

1.0 mg/mL RT b , 1 day 1166 16 102

1.0 mg/mL RT, 2 days 1167 14 102

1.0 mg/mL RT, 3days 1165 24 102

5.0 mg/mL 0 day 5743 533

-

5.0 mg/mL Ca. 4 o C, 1 day 5624 83 97.9

5.0 mg/mL RT, 1 day 5711 102 99.4

5.0 mg/mL RT, 2 days 5539 167 96.6

5.0 mg/mL RT, 3days 5250 106 91.6

a Calculated as a percentage of the day 0 value.

b RT = Room TemperatureRTI Project No.: 0209 4 08.00 7 Protocol No. RTI

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- 4 Table 2 Dose formulation analysis for high dose male rats.

Sample ID Name RT Area Dilution Amount Units Calibration

Id

1 12307

- 61

- 1 ETBE 4.052 2196 10.46000 5283.001  g/mL 5249

2 12307

- 61

- 1 ETBE 4

. 050 2126 10.46000 5111.165  g/mL 5249

3 12307

- 61

- 2 ETBE 4

. 049 2215 10.42000 5309.806  g/mL 5249

4 12307

- 61

- 2 ETBE 4

. 050 2213 10.42000 5304.189  g/mL 5249

5 12307

- 61

- 3 ETBE 4

. 050 2195 10.51000 5304.712  g/mL 5249

6 12307

- 61

- 3 ETBE 4

. 050 2120 10.51000 5119.573  g/mL 5249

Mean 5238.8

Std.

Dev. 96.0

%RSD 1.8RTI Project No.: 0209 4 08.00 7 Protocol No. RTI

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- 5 Table 3. Dose formulation analysis for high dose female rats.

Sample ID Name RT Area Dilution Amount Units Calibration Id

1 12307

- 64

- 1 ETBE 4.051 2295 10.22000 5366.492  g/ml 5900

2 12307

- 64

- 1 ETBE 4.052 2230 10.22000 5213.326  g/ml 5900

3 12307

- 64

- 2 ETBE 4.052 2143 10.78000 5282.676  g/ml 5900

4 12307

- 64

- 2 ETBE 4.051 2181 10.78000 5377.548  g/ml 5900

5 12307

- 64

- 3 ETBE 4.050 2161 10.58000 5227.800  g/ml 5900

6 12307

- 64

- 3 ETBE 4.052 2169 10.58000 5247.629  g/ml 5900

Mean 5285.9

Std. Dev. 70.7

%RSD 1.3RTI Project No.: 0209 4 08.00 7 Protocol No. RTI

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- 6 Table 4. Dose formulation analysis for low dose female rats.

Sample ID Name RT Area Dilution Amount Units Calibration Id

1 12307

- 81

- 1 ETBE 4.053 468 9.78000 1152.140  g/mL 5666

2 12307

- 81

- 1 ETBE 4

. 051 471 918000 1158.194  g/mL 5666

3 12307

- 81

- 2 ETBE 4

. 052 426 10.56000 1143.368  g/mL 5666

4 12307

- 81

- 2 ETBE 4

. 051 428 10.56000 1148.662  g/mL 5666

5 12307

- 81

- 3 ETBE 4

. 049 468 9.72000 1146.016  g/mL 5666

6 12307

- 81

- 3 ETBE 4

. 050 467 9.72000 1142.978  g/mL 5666

Mean 1148.6

Std. Dev. 5.8

%RSD 0.5RTI Project No.: 0209 4 08.00 7 Protocol No. RTI

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- 7 Table 5. Dose formulation analysis for low dose female rats.

Sample ID Name RT Area Dilution Amount Units Calibration Id

1 12307

- 84

- 1 ETBE 4.052 443 10.44000 986.780  g/mL 5830

2 12307

- 84

- 1 ETBE 4

. 053 444 10.44000 989.869  g/mL 5830

3 12307

- 84

- 2 ETBE 4

. 053 444 10.61000 1005.417  g/mL 5830

4 12307

- 84

- 2 ETBE 4

. 052 433 10.61000 981.594  g/mL 5830

5 12307

- 84

- 3 ETBE 4

. 052 423 10.97000 989.709  g/mL 5830

6 12307

- 84

- 3 ETBE 4

. 053 422 10.97000 987.555  g/mL 5830

Mean 990.2

Std. Dev. 8.1

%RSD 0.8RTI Project No.: 0209 4 08.00 7 Protocol No. RTI

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- 8RTI Project No.: 0209 4 08.00 7 Protocol No. RTI

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- 9 Figure 1. Calibration Report 5249 for Analysis of ETBE Dose Formulation.RTI Project No.: 0209 4 08.00 7 Protocol No. RTI

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- 10 Figure 1 (continued). Calibration Report 5249 for Analysis of ETBE Dose Formulation.RTI Project No.: 0209 4 08.00 7 Protocol No. RTI

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- 11 Figure 1 (continued). Calibration Report 5249 for Analysis of ETBE Dose Formulation.RTI Project No.: 0209 4 08.00 7 Protocol No. RTI

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- 12 Figure 2. Calibratio n Report 5900 for Analysis of ETBE Dose Formulation.RTI Project No.: 0209 4 08.00 7 Protocol No. RTI

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- 13 Figure 2 (continued). Calibration Report 5900 for Analysis of ETBE Dose Formulation.RTI Project No.: 0209 4 08.00 7 Protocol No. RTI

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- 14 Figure 2 (continued). Calibration Report 5900 for Analysis of ETBE Dose Formulation.RTI Project No.: 0209 4 08.00 7 Protocol No. RTI

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- 15 Figure 3. Calibration Report 5666 for Analysis of ETBE Dose Formulation.RTI Project No.: 0209 4 08.00 7 Protocol No. RTI

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- 16 Figure 3 (continued). Calibration Report 5666 for Analysis of ETBE Dose Formulation.RTI Project No.: 0209 4 08.00 7 Protocol No. RTI

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- 17 Figure 3 (continued). Calibration Report 5666 for Analysis of ETBE Dose Formulation.RTI Project No.: 0209 4 08.00 7 Protocol No. RTI

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- 18 Figure 4. Calibration Report 5830 for Analysis of ETBE Dose Formulation.RTI Project No.: 0209 4 08.00 7 Protocol No. RTI

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- 19 Figure 4 (continued) . Calibration Report 5830 for Analysis of ETBE Dose Formulation.RTI Project No.: 0209 4 08.00 7 Protocol No. RTI

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- 20 Figure 4 (continued). Calibration Report 5830 for Analysis of ETBE Dose Formulation.RTI Project No.: 0209 4 08.00 7 Protocol No. RTI

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- 21 Project Specific Analytical Method for Analysis of Ethyl Tertiary Butyl Ether in Oral Dose

FormulationsRTI Project No.: 0209 4 08.00 7 Protocol No. RTI

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- 1 Appendix F

Blood Sample Collection and Analysis for ETBERTI Project No.: 0209 4 08.00 7 Protocol No. RTI

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- 2 Table 1. Blood Sample Collection Times.

Subject Sample Dose Time Nominal

Time Elapsed Time

(hh:mm:ss) Capture Time

AM01 Blood 6/13/2007 10:03 15 m 0:15:36 6/13/2007 10:19

AM02 Blood 6/13/2007 10:10 15 m 0:14:19 6/13/2007 10:24

AM03 Blood 6/13/2007 10:18 15 m 0:14:09 6/13/2007 10:32

AM04 Blood 6/13/2007 10:25 15 m 0:14:11 6/13/2007 10:39

AM01 Blood 6/13/2007 10:03 30 m 0:30:44 6/13/2007 10:34

AM02 Blood 6/13/2007 10:10 30 m 0:29:25 6/13/2007 10:39

AM03 Blood 6/13/2007 10:18 30 m 0:29:42 6/13/2007 10:48

AM04 Blood 6/13/2007 10:25 30 m 0:30:39 6/13/2007 10:56

AM01 Blood 6/13/2007 10:03 1 h 0:59:06 6/13/2007 11:02

AM02 Blood 6/13/2007 10:10 1 h 0:58:58 6/13/2007 11:09

AM03 Blood 6/13/2007 10:18 1 h 0:59:38 6/13/2007 11:18

AM04 Blood 6/13/2007 10:25 1 h 1:01:04 6/13/2007 11:26

AM01 Blood 6/13/2007 10:03 2 h 2:03:09 6/13/2007 12:06

AM02 Blood 6/13/2007 10:10 2 h 1:59:01 6/13/2007 12:09

AM03 Blood 6/13/2007 10:18 2 h 1:59:11 6/13/2007 12:17

AM04 Blood 6/13/2007 10:25 2 h 1:59:24 6/13/2007 12:25

AM01 Blood 6/13/2007 10:03 4 h 4:03:13 6/13/2007 14:06

AM02 Blood 6/13/2007 10:10 4 h 3:58:28 6/13/2007 14:08

AM03 Blood 6/13/2007 10:18 4 h 3:58:58 6/13/2007 14:17

AM04 Blood 6/13/2007 10:25 4 h 4:00:31 6/13/2007 14:26

AM01 Blood 6/13/2007 10:03 8 h 8:07:00 6/13/2007 18:10

AM02 Blood 6/13/2007 10:10 8 h 8:02:06 6/13/2007 18:12

AM03 Blood 6/13/2007 10:18 8 h 7:59:19 6/13/2007 18:17

AM04 Blood 6/13/2007 10:25 8 h 8:00:11 6/13/2007 18:25

AM01 Blood 6/13/2007 10:03 24 h 24:09:52 6/14/2007 10:13

AM02 Blood 6/13/2007 10:10 24 h 24:09:20 6/14/2007 10:19

AM03 Blood 6/13/2007 10:18 24 h 24:06:03 6/14/2007 10:24

AM04 Blood 6/13/2007 10:25 24 h 24:03:00 6/14/2007 10:28

BF01 Blood 6/14/2007 11:13 15 m 0:13:48 6/14/2007 11:27

BF01 Blood 6/14/2007 11:13 30 m 0:28:24 6/14/2007 11:41

BF01 Blood 6/14/2007 11:13 1 h 0:59:05 6/14/2007 12:12

BF02 Blood 6/14/2007 11:18 15 m 0:17:12 6/14/2007 11:35

BF02 Blood 6/14/2007 11:18 30 m 0:28:04 6/14/2007 11:46

BF02 Blood 6/14/2007 11:18 1 h 0:58:45 6/14/2007 12:16

BF03 Blood 6/14/2007 11:27 15 m 0:12:04 6/14/2007 11:40

BF03 Blood 6/14/2007 11:27 30 m 0:29:13 6/14/2007 11:57

BF03 Blood 6/14/2007 11:27 1 h 1:00:51 6/14/2007 12:28

Table 1 (continued). Blood Sample Collection Times.

Subject Sample Dose Time Nominal Elapsed Time Capture TimeRTI Project No.: 0209 4 08.00 7 Protocol No. RTI

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- 3 Time (hh:mm:ss)

BF04 Blood 6/14/2007 11:37 15 m 0:13:18 6/14/2007 11:50

BF04 Blood 6/14/2007 11:37 30 m 0:29:49 6/14/2007 12:06

BF04 Blood 6/14/2007 11:37 1 h 0:59:21 6/14/2007 12:36

BF01 Blood 6/14/2007 11:13 2 h 1:58:43 6/14/2007 13:12

BF02 Blood 6/14/2007 11:18 2 h 1:59:03 6/14/2007 13:17

BF03 Blood 6/14/2007 11:27 2 h 2:01:56 6/14/2007 13:29

BF04 Blood 6/14/2007 11:37 2 h 1:59:31 6/14/2007 13:36

BF01 Blood 6/14/2007 11:13 4 h 4:03:52 6/14/2007 15:17

BF02 Blood 6/14/2007 11:18 4 h 4:02:44 6/14/2007 15:20

BF03 Blood 6/14/2007 11:27 4 h 4:12:40 6/14/2007 15:40

BF04 Blood 6/14/2007 11:37 4 h 4:01:30 6/14/2007 15:38

BF01 Blood 6/14/2007 11:13 8 h 8:00:00 6/14/2007 19:13

BF02 Blood 6/14/2007 11:18 8 h 7:59:52 6/14/2007 19:17

BF03 Blood 6/14/2007 11:27 8 h 8:04:35 6/14/2007 19:32

BF04 Blood 6/14/2007 11:37 8 h 8:00:57 6/14/2007 19:38

BF01 Blood 6/14/2007 11:13 24 h 24:02:42 6/15/2007 11:16

BF02 Blood 6/14/2007 11:18 24 h 24:00:12 6/15/2007 11:18

BF03 Blood 6/14/2007 11:27 24 h 24:02:32 6/15/2007 11:30

BF04 Blood 6/14/2007 11:37 24 h 24:00:56 6/15/2007 11:38

CM01 Blood 6/20/2007 9:36 15 m 0:14:12 6/20/2007 9:50

CM02 Blood 6/20/2007 9:40 15 m 0:14:18 6/20/2007 9:55

CM03 Blood 6/20/2007 9:44 15 m 0:14:17 6/20/2007 9:58

CM04 Blood 6/20/2007 9:48 15 m 0:14:34 6/20/2007 10:03

CM01 Blood 6/20/2007 9:36 30 m 0:29:45 6/20/2007 10:06

CM02 Blood 6/20/2007 9:40 30 m 0:29:26 6/20/2007 10:10

CM03 Blood 6/20/2007 9:44 30 m 0:29:35 6/20/2007 10:14

CM04 Blood 6/20/2007 9:48 30 m 0:29:35 6/20/2007 10:18

CM01 Blood 6/20/2007 9:36 1 h 0:59:30 6/20/2007 10:35

CM02 Blood 6/20/2007 9:40 1 h 0:59:43 6/20/2007 10:40

CM03 Blood 6/20/2007 9:44 1 h 0:59:36 6/20/2007 10:44

CM04 Blood 6/20/2007 9:48 1 h 0:59:46 6/20/2007 10:48

CM08 Blood 6/20/2007 10:37 15 m 0:16:24 6/20/2007 10:53

CM08 Blood 6/20/2007 10:37 30 m 0:31:34 6/20/2007 11:08

CM01 Blood 6/20/2007 9:36 2 h 1:59:06 6/20/2007 11:35

CM02 Blood 6/20/2007 9:40 2 h 2:02:08 6/20/2007 11:42

CM03 Blood 6/20/2007 9:44 2 h 2:01:48 6/20/2007 11:46

CM08 Blood 6/20/2007 10:37 2 h 1:59:46 6/20/2007 12:36

CM01 Blood 6/20/2007 9:36 4 h 3:59:41 6/20/2007 13:35

CM02 Blood 6/20/2007 9:40 4 h 4:01:04 6/20/2007 13:41

CM03 Blood 6/20/2007 9:44 4 h 3:59:29 6/20/2007 13:43

CM08 Blood 6/20/2007 10:37 4 h 3:59:39 6/20/2007 14:36

Table 1 (continued). Blood Sample Collection Times.

Subject Sample Dose Time Nominal Elapsed Time Capture TimeRTI Project No.: 0209 4 08.00 7 Protocol No. RTI

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- 4 Time (hh:mm:ss)

CM01 Blood 6/20/2007 9:36 8 h 8:03:04 6/20/2007 17:39

CM02 Blood 6/20/2007 9:40 8 h 8:01:17 6/20/2007 17:42

CM03 Blood 6/20/2007 9:44 8 h 7:59:19 6/20/2007 17:43

CM08 Blood 6/20/2007 10:37 8 h 8:01:21 6/20/2007 18:38

CM01 Blood 6/20/2007 9:36 24 h 23:59:48 6/21/2007 9:36

CM02 Blood 6/20/2007 9:40 24 h 24:03:54 6/21/2007 9:44

CM03 Blood 6/20/2007 9:44 24 h 24:01:51 6/21/2007 9:46

CM08 Blood 6/20/2007 10:37 24 h 24:01:00 6/21/2007 10:38

CM08 Blood 6/20/2007 10:37 1 h 0:59:31 6/20/2007 11:36

DF01 Blood 6/21/2007 10:04 15 m 0:14:37 6/21/2007 10:19

DF02 Blood 6/21/2007 10:11 15 m 0:14:39 6/21/2007 10:25

DF04 Blood 6/21/2007 10:34 15 m 0:17:02 6/21/2007 10:51

DF01 Blood 6/21/2007 10:04 30 m 0:30:05 6/21/2007 10:34

DF02 Blood 6/21/2007 10:11 30 m 0:28:34 6/21/2007 10:39

DF04 Blood 6/21/2007 10:34 30 m 0:29:53 6/21/2007 11:04

DF01 Blood 6/21/2007 10:04 1 h 0:58:56 6/21/2007 11:03

DF02 Blood 6/21/2007 10:11 1 h 0:58:42 6/21/2007 11:09

DF04 Blood 6/21/2007 10:34 1 h 0:58:20 6/21/2007 11:33

DF05 Blood 6/21/2007 11:02 15 m 0:23:51 6/21/2007 11:26

DF05 Blood 6/21/2007 11:02 30 m 0:29:27 6/21/2007 11:31

DF05 Blood 6/21/2007 11:02 1 h 0:57:06 6/21/2007 11:59

DF01 Blood 6/21/2007 10:04 2 h 1:59:21 6/21/2007 12:03

DF02 Blood 6/21/2007 10:11 2 h 2:01:02 6/21/2007 12:12

DF04 Blood 6/21/2007 10:34 2 h 2:01:54 6/21/2007 12:36

DF05 Blood 6/21/2007 11:02 2 h 2:00:50 6/21/2007 13:03

DF01 Blood 6/21/2007 10:04 4 h 3:58:28 6/21/2007 14:03

DF02 Blood 6/21/2007 10:11 4 h 4:00:31 6/21/2007 14:11

DF04 Blood 6/21/2007 10:34 4 h 3:43:14 6/21/2007 14:18

DF05 Blood 6/21/2007 11:02 4 h 4:00:04 6/21/2007 15:02

DF01 Blood 6/21/2007 10:04 8 h 8:00:11 6/21/2007 18:04

DF02 Blood 6/21/2007 10:11 8 h 7:58:18 6/21/2007 18:09

DF04 Blood 6/21/2007 10:34 8 h 7:58:53 6/21/2007 18:33

DF05 Blood 6/21/2007 11:02 8 h 8:08:21 6/21/2007 19:10

DF01 Blood 6/21/2007 10:04 24 h 23:59:27 6/22/2007 10:04

DF02 Blood 6/21/2007 10:11 24 h 24:07:07 6/22/2007 10:18

DF04 Blood 6/21/2007 10:34 24 h 24:00:45 6/22/2007 10:35

DF05 Blood 6/21/2007 11:02 24 h 24:01:44 6/22/2007 11:04

Table 2. Blood Sample Weights from Group A

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- 5 Subject Sample Time Pot wt Sample wt Corrected

Sample Weight

AM01 Blood Predose 11.4212 g 11.6353 g 0.2141 g

AM01 Blood 15 m 11.1270 g 11.2833 g 0.1563 g

AM01 Blood 30 m 11.1523 g 11.3527 g 0.2004 g

AM01 Blood 1 h 11.1092 g 11.2900 g 0.1808 g

AM01 Blood 2 h 11.1053 g 11.2445 g 0.1392 g

AM01 Blood 4 h 11.4308 g 11.6115 g 0.1807 g

AM01 Blood 8 h 11.4329 g 11.5216 g 0.0887 g

AM01 Blood 24 h 11.1190 g 11.2360 g 0.1170 g

AM02 Blood Predose 11.4371 g 11.6335 g 0.1964 g

AM02 Blood 15 m 11.3950 g 11.5758 g 0.1808 g

AM02 Blood 30 m 11.4324 g 11.6002 g 0.1678 g

AM02 Blood 1 h 11.1433 g 11.3230 g 0.1797 g

AM02 Blood 2 h 11.1615 g 11.3014 g 0.1399 g

AM02 Blood 4 h 11.4348 g 11.6017 g 0.1669 g

AM02 Blood 8 h 11.4201 g 11.5906 g 0.1705 g

AM02 Blood 24 h 11.2571 g 11.3627 g 0.1056 g

AM03 Blood Predose 11.1365 g 11.3031 g 0.1666 g

AM03 Blood 15 m 11.1674 g 11.3408 g 0.1734 g

AM03 Blood 30 m 11.1094 g 11.3016 g 0.1922 g

AM03 Blood 1 h 11.4141 g 11.5909 g 0.1768 g

AM03 Blood 2 h 11.4064 g 11.5602 g 0.1538 g

AM03 Blood 4 h 11.1139 g 11.3116 g 0.1977 g

AM03 Blood 8 h 11.4673 g 11.6316 g 0.1643 g

AM03 Blood 24 h 11.1560 g 11.2571 g 0.1011 g

AM04 Blood Predose 11.4306 g 11.5854 g 0.1548 g

AM04 Blood 15 m 11.1558 g 11.3193 g 0.1635 g

AM04 Blood 30 m 11.4293 g 11.5988 g 0.1695 g

AM04 Blood 1 h 11.4757 g 11.6363 g 0.1606 g

AM04 Blood 2 h 11.0899 g 11.2658 g 0.1759 g

AM04 Blood 4 h 11.1390 g 11.3385 g 0.1995 g

AM04 Blood 8 h 11.1518 g 11.3606 g 0.2088 g

AM04 Blood 24 h 11.3721 g 11.4938 g 0.1217 gRTI Project No.: 0209 4 08.00 7 Protocol No. RTI

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- 6 Table 3 . Blood Sample Weights from Group B

.

Subject Sample Time Pot wt Sample wt Corrected

Sample Weight

BF01 Blood Predose 11.1488 g 11.2307 g 0.0819 g

BF01 Blood 15 m 11.2873 g 11.4526 g 0.1653 g

BF01 Blood 30 m 11.0774 g 11.2678 g 0.1904 g

BF01 Blood 1 h 11.0936 g 11.2735 g 0.1799 g

BF01 Blood 2 h 11.3922 g 11.5856 g 0.1934 g

BF01 Blood 4 h 11.2622 g 11.4151 g 0.1529 g

BF01 Blood 8 h 11.0922 g 11.1995 g 0.1073 g

BF01 Blood 24 h 11.1848 g 11.3325 g 0.1477 g

BF02 Blood Predose 11.1217 g 11.3399 g 0.2182 g

BF02 Blood 15 m 11.3030 g 11.4662 g 0.1632 g

BF02 Blood 30 m 11.2971 g 11.4624 g 0.1653 g

BF02 Blood 1 h 11.2922 g 11.4724 g 0.1802 g

BF02 Blood 2 h 11.3644 g 11.5423 g 0.1779 g

BF02 Blood 4 h 11.0763 g 11.2486 g 0.1723 g

BF02 Blood 8 h 11.3128 g 11.4617 g 0.1489 g

BF02 Blood 24 h 11.0841 g 11.2386 g 0.1545 g

BF03 Blood Predose 11.3445 g 11.5136 g 0.1691 g

BF03 Blood 15 m 11.1272 g 11.2720 g 0.1448 g

BF03 Blood 30 m 11.0637 g 11.2508 g 0.1871 g

BF03 Blood 1 h 11.1027 g 11.2808 g 0.1781 g

BF03 Blood 2 h 11.1160 g 11.2066 g 0.0906 g

BF03 Blood 4 h 11.2468 g 11.3505 g 0.1037 g

BF03 Blood 8 h 11.0763 g 11.1955 g 0.1192 g

BF03 Blood 24 h 11.1114 g 11.2460 g 0.1346 g

BF04 Blood Predose 11.1544 g 11.3162 g 0.1618 g

BF04 Blood 15 m 11.0889 g 11.2715 g 0.1826 g

BF04 Blood 30 m 11.3214 g 11.4185 g 0.0971 g

BF04 Blood 1 h 11.0806 g 11.2183 g 0.1377 g

BF04 Blood 2 h 11.1596 g 11.3377 g 0.1781 g

BF04 Blood 4 h 11.3140 g 11.4553 g 0.1413 g

BF04 Blood 8 h 11.2672 g 11.4080 g 0.1408 g

BF04 Blood 24 h 11.1306 g 11.2838 g 0.1532 gRTI Project No.: 0209 4 08.00 7 Protocol No. RTI

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- 7 Table 4 . Blood Sample Weights from Group C

.

Subject Sample Time Pot wt Sample wt Corrected

Sample Weight

CM01 Blood Predose 11.3409 g 11.4943 g 0.1534 g

CM01 Blood 15 m 11.1423 g 11.2985 g 0.1562 g

CM01 Blood 30 m 11.3269 g 11.4757 g 0.1488 g

CM01 Blood 1 h 11.2967 g 11.4562 g 0.1595 g

CM01 Blood 2 h 11.4177 g 11.5398 g 0.1221 g

CM01 Blood 4 h 11.3391 g 11.4876 g 0.1485 g

CM01 Blood 8 h 11.3190 g 11.4859 g 0.1669 g

CM01 Blood 24 h 11.1246 g 11.3059 g 0.1813 g

CM02 Blood Predose 11.4180 g 11.5603 g 0.1423 g

CM02 Blood 15 m 11.2860 g 11.4204 g 0.1344 g

CM02 Blood 30 m 11.3723 g 11.5325 g 0.1602 g

CM02 Blood 1 h 11.2280 g 11.3301 g 0.1021 g

CM02 Blood 2 h 11.3617 g 11.4798 g 0.1181 g

CM02 Blood 4 h 11.1876 g 11.2883 g 0.1007 g

CM02 Blood 8 h 11.2039 g 11.3695 g 0.1656 g

CM02 Blood 24 h 11.3990 g 11.5725 g 0.1735 g

CM03 Blood Predose 11.2254 g 11.3787 g 0.1533 g

CM03 Blood 15 m 11.2436 g 11.4241 g 0.1805 g

CM03 Blood 30 m 11.3924 g 11.4825 g 0.0901 g

CM03 Blood 1 h 11.2066 g 11.3237 g 0.1171 g

CM03 Blood 2 h 11.2775 g 11.4064 g 0.1289 g

CM03 Blood 4 h 11.3050 g 11.4452 g 0.1402 g

CM03 Blood 8 h 11.2963 g 11.4470 g 0.1507 g

CM03 Blood 24 h 11.3003 g 11.4514 g 0.1511 g

CM08 Blood Predose 11.4752 g 11.5913 g 0.1161 g

CM08 Blood 15 m 11.4486 g 11.5910 g 0.1424 g

CM08 Blood 30 m 11.1118 g 11.2350 g 0.1232 g

CM08 Blood 1 h 11.1109 g 11.2690 g 0.1581 g

CM08 Blood 2 h 11.4776 g 11.6386 g 0.1610 g

CM08 Blood 4 h 11.4341 g 11.5507 g 0.1166 g

CM08 Blood 8 h 11.4683 g 11.6235 g 0.1552 g

CM08 Blood 24 h 11.4605 g 11.7199 g 0.2594 gRTI Project No.: 0209 4 08.00 7 Protocol No. RTI

- 93 2 Final Report

F

- 8 Table 4 . Blood Sample Weights from Group D

.

Subject Sample Time Pot wt Sample wt Corrected

Sample Weight

DF01 Blood Predose 11.4138 g 11.5602 g 0.1464 g

DF01 Blood 15 m 11.2374 g 11.3676 g 0.1302 g

DF01 Blood 30 m 11.1363 g 11.2859 g 0.1496 g

DF01 Blood 1 h 11.3407 g 11.5101 g 0.1694 g

DF01 Blood 2 h 11.1892 g 11.3090 g 0.1198 g

DF01 Blood 4 h 11.1345 g 11.2604 g 0.1259 g

DF01 Blood 8 h 11.4590 g 11.6008 g 0.1418 g

DF01 Blood 24 h 11.2978 g 11.4413 g 0.1435 g

DF02 Blood Predose 11.3077 g 11.4628 g 0.1551 g

DF02 Blood 15 m 11.3979 g 11.5384 g 0.1405 g

DF02 Blood 30 m 11.3715 g 11.5452 g 0.1737 g

DF02 Blood 1 h 11.1177 g 11.2308 g 0.1131 g

DF02 Blood 2 h 11.3159 g 11.4197 g 0.1038 g

DF02 Blood 4 h 11.4005 g 11.4837 g 0.0832 g

DF02 Blood 8 h 11.1653 g 11.3037 g 0.1384 g

DF02 Blood 24 h 11.2631 g 11.4267 g 0.1636 g

DF04 Blood Predose 11.1653 g 11.2854 g 0.1201 g

DF04 Blood 15 m 11.2471 g 11.3944 g 0.1473 g

DF04 Blood 30 m 11.1474 g 11.2523 g 0.1049 g

DF04 Blood 1 h 11.3201 g 11.4407 g 0.1206 g

DF04 Blood 2 h 11.1649 g 11.2995 g 0.1346 g

DF04 Blood 4 h 11.1617 g 11.2531 g 0.0914 g

DF04 Blood 8 h 11.1842 g 11.2963 g 0.1121 g

DF04 Blood 24 h 11.1453 g 11.3038 g 0.1585 g

DF05 Blood Predose 11.4396 g 11.5576 g 0.1180 g

DF05 Blood 15 m 11.3806 g 11.4930 g 0.1124 g

DF05 Blood 30 m 11.1289 g 11.1910 g 0.0621 g

DF05 Blood 1 h 11.1786 g 11.3300 g 0.1514 g

DF05 Blood 2 h 11.3245 g 11.3913 g 0.0668 g

DF05 Blood 4 h 11.4156 g 11.5363 g 0.1207 g

DF05 Blood 8 h 11.3797 g 11.5310 g 0.1513 g

DF05 Blood 24 h 11.1243 g 11.2416 g 0.1173 gRTI Project No.: 0209 4 08.00 7 Protocol No. RTI

- 93 2 Final Report

F

- 9 Table 5. Low Concentration Calibration Curve for High Dose Groups A and B.

Peak Area

Sample I.D. Standard I.D. Conc.

(  g/ml) ETBE MTBE Peak Area

Ratio Cal. Conc.

(  g/ml) Mean

(  g/mL) Accuracy

(%) Error

(%)

12307

- 56A Blood blank n/a 0 n/a n/a n/a n/a

- -

12307

- 57A Blood blank n/a 0 n/a n/a n/a

- - -

12307

- 58A Blood blank n/a 0 n/a n/a n/a

- - -

12307

- 56B Blood ISTD blank n/a 0 2641319 n/a n/a n/a

- -

12307

- 57A Blood ISTD blank n/a 0 2154647 n/a n/a

- - -

12307

- 58A Blood ISTD blank n/a 0 2302458 n/a n/a

- - -

12307

- 56C Std A1 0.102 22552 2602098 0.00867 0.103 0.104 101% 1.43%

12307

- 57C Std A1 0.102 18713 2138062 0.00875 0.104

- 102% 2.45%

12307

- 58C Std A1 0.102 19885 2275009 0.00874 0.104

- 102% 2.31%

12307

- 56D Std B1 0.252 52855 2554845 0.0207 0.249 0.253 98.8%

- 1.25%

12307

- 57D Std B1 0.252 44467 2107229 0.0211 0.254

- 101% 0.739%

12307

- 58D Std B1 0.252 46168 2172972 0.0212 0.256

- 101% 1.43%

12307

- 56E Std A2 0.497 102237 2562139 0.0399 0.481 0.485 96.8%

- 3.17%

12307

- 57E Std A2 0.497 85024 2102020 0.0404 0.488

- 98.2%

- 1.84%

12307

- 57E Std A2 0.497 90406 2243675 0.0403 0.486

- 97.8%

- 2.22%

12307

- 56F Std B2 1.03 209164 2505428 0.0835 1.01 1.03 97.9%

- 2.10%

12307

- 57F Std B2 1.03 182555 2092192 0.0873 1.05

- 102% 2.33%

12307

- 58F Std B2 1.03 188676 2215712 0.0852 1.03

- 99.9%

- 0.14%

12307

- 56G Std A3 2.43 495521 2476881 0.200 2.42 2.43 99.5%

- 0.481%

12307

- 57G Std A3 2.43 431537 2116191 0.204 2.47

- 101% 1.44%

12307

- 57G Std A3 2.43 445943 2249957 0.198 2.40

- 98.6%

- 1.41%

12307

- 56H Std B3 5.17 1046174 2490883 0.420 5.08 5.18 98.2%

- 1.77%

12307

- 57H Std B3 5.17 922001 2129104 0.433 5.24

- 101% 1.28%

12307

- 58H Std B3 5.17 952742 2199941 0.433 5.24

- 101% 1.29%RTI Project No.: 0209 4 08.00 7 Protocol No. RTI

- 93 2 Final Report

F

- 10 Table 5 (continued). Low Concentration Calibration Curve for High Dose Groups A and B.

Peak Area

Sample I.D. Standard I.D. Conc.

(  g/ml) ETBE MTBE Peak Area

Ratio Cal. Conc.

(  g/ml) Mean

(  g/mL) Accuracy

(%) Error

(%)

12307

- 65

- 1 LQC 0.497 101327 2426211 0.0418 0.504

- 101% 1.36%

12307

- 65

- 2 LQC 0.497 98466 2404023 0.0410 0.494

- 99.4%

- 0.598%

12307

- 65

- 1 MQC 5.17 1049759 2348383 0.4470 5.41

- 105% 4.55%

12307

- 65

- 2 MQC 5.17 965608 2252523 0.4287 5.18

- 100% 0.260%

a INTERCEPT 0.0001127

b SLOPE 0.08268

r 0.9998RTI Project No.: 0209 4 08.00 7 Protocol No. RTI

- 93 2 Final Report

F

- 11 Table 6. High Concentration Calibration Curve for High Dose Groups A and B.

Peak Area

Sample I.D. Standard I.D. Conc.

(  g/ml) ETBE MTBE Peak Area

Ratio Cal. Conc.

(  g/ml) Mean

(  g/mL) Accuracy

(%) Error

(%)

12307

- 56H Std B3 5.17 1046174 2490883 0.420 5.45 5.54 105% 5.41%

12307

- 57H Std B3 5.17 922001 2129104 0.433 5.58

- 108% 8.01%

12307

- 58H Std B3 5.17 952742 2199941 0.433 5.58

- 108% 8.02%

12307

- 56I Std A4 9.76 2055076 2477991 0.829 9.66 9.74 99.0%

- 1.031%

12307

- 57I Std A4 9.76 1801497 2108315 0.854 9.92

- 102% 1.62%

12307

- 58I Std A4 9.76 1797962 2175731 0.826 9.63

- 98.7%

- 1.34%

12307

- 56J Std B4 25.8 5505071 2415755 2.28 24.6 24.9 95.2%

- 4.78%

12307

- 57J Std B4 25.8 4901307 2117403 2.31 24.9

- 96.6%

- 3.35%

12307

- 58J Std B4 25.8 5180410 2223813 2.33 25.1

- 97.2%

- 2.76%

12307

- 56K Std A5 48.9 10630375 2384396 4.46 47.0 47.5 96.1%

- 3.93%

12307

- 57K Std A5 48.9 9413503 2073300 4.54 47.8

- 97.8%

- 2.20%

12307

- 58K Std A5 48.9 9961182 2193738 4.54 47.8

- 97.8%

- 2.19%

12307

- 56L Std B5 104 22891077 2357577 9.71 101 102 96.7%

- 3.27%

12307

- 57L Std B5 104 20673056 2090026 9.89 103

- 98.5%

- 1.48%

12307

- 58L Std B5 104 20351677 2078699 9.79 102

- 97.5%

- 2.48%

12307

- 56M Std A6 249 54977181 2292603 24.0 248 253 99.6%

- 0.386%

12307

- 57M Std A6 249 50642541 2039543 24.8 256

- 103% 3.13%

12307

- 58M Std A6 249 52411517 2118717 24.7 256

- 103% 2.74%

12307

- 63

- 1 HQC 104 22085053 2166264 10.2 106

- 102% 1.51%

12307

- 63

- 1 HQC 104 24612373 2365010 10.4 108

- 104% 3.60%

a INTERCEPT

- 0.10995

b SLOPE 0.09724

r 0.9996RTI Project No.: 0209 4 08.00 7 Protocol No. RTI

- 93 2 Final Report

F

- 12 Table 7. ETBE Blood Concentration Analysis for Groups A and B.

Peak Area

Rat. Time

point ETBE MTBE Peak Area

Ratio Cal. Conc.

(  g/ml) Sample

weight (g) Corrected

Conc.

(  g/ml)

AM 01 Predose 0 2196830 0 < LOQ 0.2141 < LOQ

15 min 1816910 2302967 0.789 9.24 0.1563 5.91

30 min 1262099 2141714 0.589 7.19 0.2004 3.59

1 hr 559988 2161316 0.259 3.13 0.1808 1.73

2 hr 138439 2121503 0.0653 0.788 0.1392 0.566

4 hr 94938 2035726 0.0466 0.563 0.1807 0.311

8 hr 3241 2199731 0.00147 < LOQ 0.0887 < LOQ

24 hr 0 2525626 0.000 < LOQ 0.1170 < LOQ

AM 02 Predose 0 2222068 0.000 < LOQ 0.1964 < LOQ

15 min 2067688 2209167 0.936 10.8 0.1808 5.95

30 min 872119 2068735 0.422 5.10 0.1678 3.04

1 hr 601965 2158669 0.279 3.37 0.1797 1.88

2 hr 153565 2103540 0.0730 0.88 0.1399 0.630

4 hr 78277 2100924 0.0373 0.45 0.1669 0.269

8hr 14205 2056302 0.00691 < LOQ 0.1705 < LOQ

24 hr 0 2482458 0.000 < LOQ 0.1056 < LOQ

AM 03 Predose 0 2203725 0.000 < LOQ 0.1666 < LOQ

15 min 2473802 2162929 1.14 12.9 0.1734 7.44

30 min 1743477 2118961 0.823 9.59 0.1922 4.99

1 hr 742683 2153754 0.345 4.17 0.1768 2.36

2 hr 181407 2064299 0.0879 1.06 0.1538 0.690

4 hr 87348 2045608 0.0427 0.515 0.1977 0.261

8 hr 11530 2123129 0.00543 < LOQ 0.1643 < LOQ

24 hr 0 2413202 0.000 < LOQ 0.1011 < LOQRTI Project No.: 0209 4 08.00 7 Protocol No. RTI

- 93 2 Final Report

F

- 13 Table 7 (continued). ETBE Blood Concentration Analysis for Groups A and B.

Peak Area

Rat. Time

point ETBE MTBE Peak Area

Ratio Cal. Conc.

(  g/ml) Sample

weight (g) Corrected

Conc.

(  g/ml)

AM 04 Predose 0 2257976 0.000 < LOQ 0.1548 < LOQ

15 min 2064431 2140584 0.964 11.0 0.1635 6.76

30 min 901525 2165470 0.416 5.03 0.1695 2.97

1 hr 395917 2158230 0.183 2.22 0.1606 1.38

2 hr 206336 2105150 0.0980 1.18 0.1759 0.673

4 hr 113612 2045057 0.0556 0.671 0.1995 0.336

8 hr 12628 2095348 0.00603 < LOQ 0.2088 < LOQ

24 hr 0 2431724 0.000 < LOQ 0.1217 < LOQ

BF 01 Predose 0 2502344 0.000 < LOQ 0.0819 < LOQ

15 min 1799456 2499526 0.720 8.53 0.1653 5.16

30 min 1446798 2217557 0.652 7.84 0.1904 4.12

1 hr 658221 2267553 0.290 3.51 0.1799 1.95

2 hr 414134 2357315 0.176 2.12 0.1934 1.10

4 hr 55978 2242089 0.0250 0.301 0.1529 0.197

8 hr 10761 2474968 0.00435 < LOQ 0.1073 < LOQ

24 hr 0 2410264 0.000 < LOQ 0.1477 < LOQ

BF 02 Predose 0 2352336 0.000 < LOQ 0.2182 < LOQ

15 min 1340572 2443639 0.549 6.77 0.1632 4.15

30 min 903103 2262860 0.399 4.83 0.1653 2.92

1 hr 744583 2255543 0.330 3.99 0.1802 2.21

2 hr 315703 2323147 0.136 1.64 0.1779 0.923

4 hr 96914 2216732 0.0437 0.527 0.1723 0.306

8 hr 12338 2409563 0.00512 < LOQ 0.1489 < LOQ

24 hr 0 2367956 0.000 < LOQ 0.1545 < LOQRTI Project No.: 0209 4 08.00 7 Protocol No. RTI

- 93 2 Final Report

F

- 14 Table 7 (continued). ETBE Blood Concentration Analysis for Groups A and B.

Peak Area

Rat. Time

point ETBE MTBE Peak Area

Ratio Cal. Conc.

(  g/ml) Sample

weight (g) Corrected

Conc.

(  g/ml)

BF 03 Predose 0 2390631 0.000 < LOQ 0.1691 < LOQ

15 min 1825330 2408157 0.758 8.93 0.1448 6.16

30 min 1107295 2230081 0.497 6.24 0.1871 3.33

1 hr 521625 2357660 0.221 2.67 0.1781 1.50

2 hr 122492 2341144 0.0523 0.63 0.0906 0.697

4 hr 65921 2205701 0.0299 0.36 0.1037 0.347

8 hr 3110 2387305 0.00130 < LOQ 0.1192 < LOQ

24 hr 0 2358860 0.000 < LOQ 0.1346 < LOQ

BF 04 Predose 0 2329569 0.000 < LOQ 0.1618 < LOQ

15 min 3015285 2341472 1.29 14.4 0.1826 7.87

30 min 749363 2305076 0.325 3.93 0.0971 4.05

1 hr 515108 2279502 0.226 2.73 0.1377 1.98

2 hr 210496 2276311 0.0925 1.12 0.1781 0.627

4 hr 48962 2155643 0.0227 0.273 0.1413 0.193

8 hr 15377 2358448 0.00652 < LOQ 0.1408 < LOQ

24 hr 0 2350284 0.000 < LOQ 0.1532 < LOQRTI Project No.: 0209 4 08.00 7 Protocol No. RTI

- 93 2 Final Report

F

- 15 Table 8. Low Concentration Calibration Curve for Low Dose Groups C and D.

Peak Area

Sample I.D. Standard I.D. Conc.

(  g/ml) ETBE MTBE Peak Area

Ratio Cal. Conc.

(  g/ml) Mean

(  g/ml) Accuracy

(%) Error

(%)

12307

- 76A Blood blank n/a 0 n/a n/a n/a n/a

- -

12307

- 77A Blood blank n/a 0 n/a n/a n/a

- - -

12307

- 78A Blood blank n/a 0 n/a n/a n/a

- - -

12307

- 76B Blood ISTD blank n/a 0 2401193 n/a n/a n/a

- -

12307

- 77A Blood ISTD blank n/a 0 2549251 n/a n/a

- - -

12307

- 78A Blood ISTD blank n/a 0 2242567 n/a n/a

- - -

12307

- 76C Std A1 0.0987 19679 2385455 0.00825 0.104 0.103 105% 5.12%

12307

- 77C Std A1 0.0987 20767 2524119 0.00823 0.103

- 105% 4.86%

12307

- 78C Std A1 0.0987 17993 2213878 0.00813 0.102

- 104% 3.67%

12307

- 76 D Std B1 0.267 52632 2369143 0.0222 0.267 0.267 100% 0.00615%

12307

- 77D Std B1 0.267 55885 2491181 0.0224 0.270

- 101% 0.958%

12307

- 78D Std B1 0.267 47302 2141561 0.0221 0.266

- 99.4%

- 0.554%

12307

- 76 E Std A2 0.500 95098 2336737 0.0407 0.483 0.481 96.6%

- 3.39%

12307

- 77E Std A2 0.500 100776 2478539 0.0407 0.483

- 96.5%

- 3.47%

12307

- 7 8 E Std A2 0.500 86099 2139232 0.0402 0.478

- 95.6%

- 4.44%

12307

- 76 F Std B2 1.08 211638 2347792 0.0901 1.06 1.06 98.3%

- 1.75%

12307

- 77F Std B2 1.08 222909 2504261 0.0890 1.05

- 97.0%

- 2.97%

12307

- 78F Std B2 1.08 201097 2192174 0.0917 1.08

- 100%

- 0.03%

12307

- 76 G Std A3 2.51 493023 2339302 0.211 2.47 2.52 98.4%

- 1.550%

12307

- 77G Std A3 2.51 531189 2456918 0.216 2.53

- 101% 0.99%

12307

- 7 8 G Std A3 2.51 471407 2168767 0.217 2.55

- 102% 1.53%

12307

- 76 H Std B3 5.36 1038513 2281832 0.455 5.33 5.39 99.4%

- 0.601%

12307

- 77H Std B3 5.36 1131632 2464067 0.459 5.38

- 100% 0.300%

12307

- 78H Std B3 5.36 1020627 2189878 0.466 5.46

- 102% 1.79%RTI Project No.: 0209 4 08.00 7 Protocol No. RTI

- 93 2 Final Report

F

- 16 Table 8 (continued). Low Concentration Calibration Curve for Low Dose Groups C and D.

Peak Area

Sample I.D. Standard I.D. Conc.

(  g/ml) ETBE MTBE Peak Area

Ratio Cal. Conc.

(  g/ml) Mean

(  g/ml) Accuracy

(%) Error

(%)

12307

- 85

- A LQC 0.500 101692 2560188 0.0397 0.472

- 94.3%

- 5.67%

12307

- 86

- A LQC 0.500 91467 2379344 0.0384 0.457

- 91.3%

- 8.66%

12307

- 85

- B MQC 5.36 1187024 2584089 0.459 5.38

- 100% 0.323%

12307

- 86

- B MQC 5.36 1006382 2309839 0.436 5.10

- 95.2%

- 4.84%

a INTERCEPT

- 0.00062549

b SLOPE 0.085542

r 0.9998RTI Project No.: 0209 4 08.00 7 Protocol No. RTI

- 93 2 Final Report

F

- 17 Table 9. High Concentration Calibration Curve for Low Dose Groups C and D.

Peak Area

Sample I.D. Standard I.D. Conc.

(  g/ml) ETBE MTBE Peak Area

Ratio Cal. Conc.

(  g/ml) Mean

(  g/mL) Accuracy

(%) Error

(%)

12307

- 76H Std B3 5.36 1038513 2281832 0.455 5.81 5.86 108% 8.46%

12307

- 77H Std B3 5.36 1131632 2464067 0.459 5.85

- 109% 9.22%

12307

- 78H Std B3 5.36 1020627 2189878 0.466 5.92

- 110% 10.5%

12307

- 76I Std A4 9.91 1966434 2295658 0.857 9.77 10.0 98.6%

- 1.394%

12307

- 77I Std A4 9.91 2171596 2470927 0.879 10.0

- 101% 0.82%

12307

- 78I Std A4 9.91 1940862 2161534 0.898 10.2

- 102.7% 2.72%

12307

- 76J Std B4 27.0 5549321 2271644 2.44 25.4 25.6 94.1%

- 5.88%

12307

- 77J Std B4 27.0 5934902 2435181 2.44 25.4

- 93.9%

- 6.09%

12307

- 78J Std B4 27.0 5399118 2157220 2.50 26.0

- 96.3%

- 3.69%

12307

- 76K Std A5 50.2 10647916 2292558 4.64 47.1 47.9 93.9%

- 6.13%

12307

- 77K Std A5 50.2 11671433 2467353 4.73 48.0

- 95.6%

- 4.45%

12307

- 78K Std A5 50.2 10276072 2139796 4.80 48.7

- 97.0%

- 3.03%

12307

- 76L Std B5 109 22795863 2190902 10.4 104 106 95.2%

- 4.84%

12307

- 77L Std B5 109 25778388 2435123 10.6 106

- 96.8%

- 3.20%

12307

- 78L Std B5 109 23794507 2191879 10.9 108

- 99.2%

- 0.766%

12307

- 76M Std A6 254 58000722 2234294 26.0 257 260 101% 1.45%

12307

- 77M Std A6 254 63467516 2444398 26.0 257

- 101% 1.47%

12307

- 78M Std A6 254 58021039 2173056 26.7 265

- 104% 4.33%

12307

- 85

- C HQC 109 23890305 2317020 10.3 103

- 94.3%

- 5.69%

12307

- 85

- D HQC 109 24163823 2385436 10.1 101

- 92.7%

- 7.32%

a INTERCEPT

- 0.13447

b SLOPE 0.10142

r 0.999RTI Project No.: 0209 4 08.00 7 Protocol No. RTI

- 93 2 Final Report

F

- 18 Table 10. ETBE Blood Concentration Analysis for Groups C and D.

Peak Area

Sample I.D. Time point ETBE MTBE Peak Area

Ratio Cal. Conc.

(  g/ml) Sample

weight (g) Corrected

Conc. (  g/ml)

CM 01 Predose 0 2708273 0 <LOQ 0.1534 <LOQ

15 min 364965 2693222 0.136 1.59 0.1562 1.02

30 min 138414 2603538 0.0532 0.629 0.1488 0.423

1 hr 73836 2526019 0.0292 0.349 0.1595 0.219

2 hr 24331 2593718 0.00938 0.117 0.1221 0.0958

4 hr 7766 2587412 0.00300 <LOQ 0.1485 <LOQ

8 hr 0 2482179 0.000 <LOQ 0.1669 <LOQ

24 hr 0 2356970 0.000 <LOQ 0.1813 <LOQ

CM 02 Predose 0 2710000 0.000 <LOQ 0.1423 <LOQ

15 min 378788 2671807 0.142 1.66 0.1344 1.24

30 min 176491 2581415 0.0684 0.807 0.1602 0.503

1 hr 47674 2557427 0.0186 0.225 0.1021 0.221

2 hr 19821 2548846 0.00778 <LOQ 0.1181 <LOQ

4 hr 6084 2576653 0.00236 <LOQ 0.1007 <LOQ

8hr 0 2437790 0.000 <LOQ 0.1656 <LOQ

24 hr 0 2397593 0.000 <LOQ 0.1735 <LOQ

CM 03 Predose 0 2586244 0.000 <LOQ 0.1533 <LOQ

15 min 454277 2600777 0.175 2.05 0.1805 1.14

30 min 104522 2516129 0.0415 0.493 0.0901 0.547

1 hr 66995 2532186 0.0265 0.317 0.1171 0.270

2 hr 17275 2535485 0.00681 <LOQ 0.1289 <LOQ

4 hr 8494 2487809 0.00341 <LOQ 0.1402 <LOQ

8 hr 0 2452134 0.0000 <LOQ 0.1507 <LOQ

24 hr 0 2457582 0.000 <LOQ 0.1511 <LOQRTI Project No.: 0209 4 08.00 7 Protocol No. RTI

- 93 2 Final Report

F

- 19 Table 10 (continued). ETBE Blood Concentration Analysis for Groups C and D.

Peak Area

Sample I.D. Time point ETBE MTBE Peak Area

Ratio Cal. Conc.

(  g/ml) Sample

weight (g) Corrected

Conc. (  g/ml)

CM 08 Predose 0 2678144 0.000 <LOQ 0.1161 <LOQ

15 min 270768 2683076 0.101 1.19 0.1424 0.834

30 min 117962 2601598 0.045 0.537 0.1232 0.436

1 hr 63368 2524024 0.0251 0.301 0.1581 0.190

2 hr 25761 2479064 0.0104 0.129 0.161 0.0800

4 hr 4883 2517248 0.0019 <LOQ 0.1166 <LOQ

8 hr 0 2439774 0.0000 <LOQ 0.1552 <LOQ

24 hr 0 2327966 0.000 <LOQ 0.2594 <LOQ

DF 01 Predose 0 2589962 0.000 <LOQ 0.1464 <LOQ

15 min 345332 2480756 0.139 1.63 0.1302 1.26

30 min 197482 2382581 0.083 0.976 0.1496 0.653

1 hr 115407 2521028 0.046 0.542 0.1694 0.320

2 hr 27345 2337992 0.0117 0.144 0.1198 0.120

4 hr 8001 2453513 0.0033 <LOQ 0.1259 <LOQ

8 hr 2106 2535169 0.00083 <LOQ 0.1418 <LOQ

24 hr 0 2367633 0.000 <LOQ 0.1435 <LOQ

DF 02 Predose 0 2510725 0.000 <LOQ 0.1551 <LOQ

15 min 297826 2381214 0.125 1.47 0.1405 1.05

30 min 196106 2398964 0.0817 0.963 0.1737 0.554

1 hr 59294 2427433 0.0244 0.293 0.1131 0.259

2 hr 19440 2478320 0.00784 <LOQ 0.1038 <LOQ

4 hr 6952 2464808 0.00282 <LOQ 0.0832 <LOQ

8 hr 0 2407469 0.00000 <LOQ 0.1384 <LOQ

24 hr 0 2296209 0.000 <LOQ 0.1636 <LOQRTI Project No.: 0209 4 08.00 7 Protocol No. RTI

- 93 2 Final Report

F

- 20 Table 10 (continued). ETBE Blood Concentration Analysis for Groups C and D.

Peak Area

Sample I.D. Time point ETBE MTBE Peak Area

Ratio Cal. Conc.

(  g/ml) Sample

weight (g) Corrected

Conc. (  g/ml)

DF 04 Predose 0 2445110 0.000 <LOQ 0.1201 <LOQ

15 min 304137 2407123 0.126 1.48 0.1473 1.01

30 min 97237 2390413 0.0407 0.483 0.1049 0.460

1 hr 58151 2356125 0.0247 0.296 0.1206 0.245

2 hr 26052 2376773 0.0110 0.135 0.1346 0.101

4 hr 11934 2406270 0.00496 <LOQ 0.0914 <LOQ

8 hr 2192 2434416 0.00090 <LOQ 0.1121 <LOQ

24 hr 0 2184403 0.000 <LOQ 0.1585 <LOQ

DF 05 Predose 0 2617559 0.000 <LOQ 0.1180 <LOQ

15 min 65880 2489293 0.0265 0.317 0.1124 0.282

30 min 39043 2483004 0.0157 0.191 0.0621 0.308

1 hr 80375 2353090 0.0342 0.407 0.1514 0.269

2 hr 10540 2351428 0.00448 <LOQ 0.0668 <LOQ

4 hr 4837 2516128 0.00192 <LOQ 0.1207 <LOQ

8 hr 0 2450106 0.00000 <LOQ 0.1513 <LOQ

24 hr 0 2257636 0.000 <LOQ 0.1173 <LOQ