March 6, 2013 Audited Final Draft Report

- ETHYL TERTIARY BUTYL ETHER ORAL ABSORPTION IN

MALE AND FEMALE RATS



### 1 COMMENTS Action / Response Page # Dr. Richard Schlesinger

1 The original goals of the study have been met. The overall conclusions are supported by the reported

results. Agree , no change

2 Why were there 4 animals in Groups A and B, and

5 in C and D. 

### No change. 5 animals were dosed in groups C and D, but one was removed from the study because the dose administered was substantially less than the target, and the other lost cannula patency and was replaced. See footnote in

Table 3. 14

3 It is stated that blood was drawn from 4 rats of each sex at various time points. So, what happened to the 5 th animal in C and D. 

### No change. See footnotes to Table 3.

4 It is stated that the t test was used and that using this test, males were compared to females within dose group for each parameter. Why did the investigators not perform statistical tests between the high and low within each sex. It seems that they

could have performed an ANOVA that would have evaluated any between gender difference at each dose as well as any between dose level difference within each gender alone. 

### T here are two completely differ ing opinions expressed by the reviewers about the

statistical analysis ranging from do all the comparisons to do none. We chose the middle ground, comparing between gender for the same dose, and between dose for the dose normalized parameter
AUCobserved.Dose. The statistical analysis was not conducted for all

para meters because of the concern that the values obtained at the low dose may have been influenced by the sensitivity of the method for analysis.

5 It was noted that no statistical differences were

noted between males and females in the pharmacokinetic par ameters measured. However,

in Table 8 the t 1⁄2 Lambda z is much lower in males than females. The SD in males for this parameter seems high compared to other groups. This requires some discussion 

### There is extensive discussion of the reason

for this difference, and the statistical analysis was conducted with and without

DF05 included in the calculation. This animal accounted for the large difference in variability. See last paragraph on page 7. No change

Dr. James BondMarch 6, 2013 Audited Final Draft Report

- ETHYL TERTIARY BUTYL ETHER ORAL ABSORPTION IN

MALE AND FEMALE RATS



### 2 1 The objectives of this study of ETBE were to: 1. Develop a method for the analysis of ETBE in blood by headspace gas chromatography

– mass spectrometry (GC

- MS). 2. Conduct an evaluation of oral absorption by measuring ETBE in blood in male and female

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

Both of these objectives appear to have been accomplished in an acceptable manner. The doses chosen for assessment of oral absorption were well justified, as was the vehicle (water) for

administration of the ETBE. Agree , no change

2 Since PK analysis of ETBE in the blood was not a stated objective of this study the final report would be significantly strengthened if information on, and conclusions derived from, the PK analysis were removed from the Summary and Discussion. More

useful information in both the Summary and Discussion would be the actual experimental data obtained in this study (e.g., mean maximum ETBE concentrations in blood for each group of rats exposed to ETBE). 

### Disag ree. The RFP for this work asked for

determination of the kinetics of ETBE. Each timepoint is a point in time. However,

the determination of the kinetics of removal and area under the curve provides an understanding of how persistent ETBE is.

3 Be cause quantitative data were obtained for a

maximum of 5 time

- points (the 8 and 24 hour samples were below the limit of quantitation), there are not sufficient experimental data to adequately describe a model containing the multiple phases following oral a dministration. In fact, it appears that the first phase, the absorption phase, may have

been missed before the first data point was collected at 0.25 hours as at this time point the

maximum blood level was obtained in all 4 exposure groups. Collection of earlier time points (e.g., 2, 5 or 10 minutes) may have resulted in lower values enabling the characterization of an absorption phase. 

### Agreed. The original design requested by

API was for 0, 4, 8, 16, 24 hours after dosing. RTI proposed adding additional timepoints earlier in the study. The revised study design provided sufficient data to demonstrate that absorption occurred and

to evaluate the elimination.March 6, 2013 Audited Final Draft Report

- ETHYL TERTIARY BUTYL ETHER ORAL ABSORPTION IN

MALE AND FEMALE RATS



### 3 4 It is also possible that the true peak in ETBE blood

concentration was not obtained in these studies as it may have occurred before 0.25 hours. Visual examination of the graphs presented in Appendix D suggests that first time

- point 0.25 hours may in fact be part of the distribution phase. Data collected at the later time points (e.g., 2 and 4 hour s) may be part of the elimination phase and the data collected at 1 hr may combine elements of distribution and elimination. Unfortunately, there are insufficient data points to adequately describe the pharmacokinetics of oral absorption. Given the complex ity of the oral absorption process and the fact that rats in this study were fitted with indwelling cannulae that typically allow for both sampling at early time points (e.g., <5 min) and rapid collection of multiple samples from the same animal, it is

unc lear why earlier and more frequent blood samples were not collected to better characterize the pharmacokinetics after oral administration of

ETBE. 

### See response to item 3 above. In light of the data collected, it is now much easier to design a better study. However, there is a limit to the number of samples that could be analyzed in the stability window, and a limit to the number of samples that could

be withdrawn from each animal. The spacing of samples was chosen to provide a balance of covering the 24 h our period, enabling the headspace analysis to be conducted, and limiting the number of samples per animal.

5 Examination of the information in Appendix D

indicates that in numerous instances all the data

collected on ETBE concentrations in blood were included in the PK analysis and in other instances some data points were eliminated. This
inconsistent inclusion/exclusion of data results in skewed analyses especially in comparisons of the

half

- times for elimination for the high and low

doses. Inclusion of all data points, including early time points likely representing the distribution phase, resultsin shorter elimination half

- times than

half

- times obtained when only later time points are

used. For example, for the high dose male group when all 5 data po ints (0.25, 0.5, 1 and 4 hr) were used in the PK analysis for 2 of the rats, the calculated half

times were 0.92 and 0.78 hr. When the 3 or 4 later time points only were used (0.25 and/or 0.5 hr were eliminated) for the other 2 rats, calculated half

times of 1.0 and 1.5 hr were obtained. Similar observations are seen for females at the high dose. 

### No change. I disagree about bias and

inconsistent inclusion or exclusion of data

. WinNonlin analysis was conducted with automated fit of the data, in which the

s oftware selects which points to include to give the best fit. This is less open to question than manually selecting the data for modeling , where operator bias could be

a factor

.March 6, 2013 Audited Final Draft Report

- ETHYL TERTIARY BUTYL ETHER ORAL ABSORPTION IN

MALE AND FEMALE RATS



### 4 6 Thus, the conclusions drawn in the Summary and

Discussion are not correct. The reported half times

for the low dose are shorter than those of the high

dose because of a bias due to inconsistencies in analysis rather than due to metabolic saturation at the high dose. Because of the small number of time points available fo r analysis and the complex, multi

phasic nature of the time course after oral administration, no conclusions can be, or should be, drawn from the PK analysis. 

### I entirely d isagree with the reviewer. The statement begins “ T hus....” Indicating that the contention is based on the previous point, which I disagree with entirely. The reviewer is inferring a more complex picture than is apparent from the data. We have no operator bias in selection of points. We are using a non

- co mpartmental

model.

7 Page 11 of 217, Section 3.6.1: Is there an SOP for blood collection in rats with jugular vein cannulae? It would seem that the methodology used to draw the blood (i.e., whether the cannula was flushed with heparin, or if blood in t he cannula was cleared to prevent dilution of the subsequent sample) would be importantinformation to included in this report. Additionally, were samples of blood takenfor analysis of hematocrit before and after the study? If not, can the authors make a comment/conclusion relative to the change in blood volume for the animal over the duration of the study? 

### A description of the blood sampling procedure has been added to the report.

After each sam ple the cannula was filled with heparinized saline. We did not sample blood for hematocrit. All blood samples were used for headspace analysis.

8 Page 12 of 217, Section 3.7: In general the usefulness of the PK analysis is somewhat questionable. This is due to the fact that the one compartment model used by the program is probably not adequate for analysis of

concentrations in blood after gavage dosing. The first blood sample taken was the highest, thus the absorption phase of the time course was miss ed,

and there is no assurance that the maximum concentration achieved in the blood during this study was actually at the first time point. Thus, reporting of Cmax and Tmax may be misleading.

Since the animals were implanted with indwelling cannulae rapid s ampling of blood at multiple early time points (e.g., 5 min, 10 min) could have been conducted. It is not clear why the investigators did not include earlier sampling or why they did not conduct a preliminary study to determine the optimal sampling times ( i.e., earlier rather than later). 

### I d isagree with the assertion concerning the utility of the PK analysis . The analysis

was not conducted with a one

compartment model. It was conducted

with a non

- compartmental analysis . It is widely used because it does not require an assumption of the number of compartments used. The non

compartmental approach is entirely appropriate for this kind of data. In fact RTI proposed earlier timepoints than were originally requested in the RFP.March 6, 2013 Audited Final Draft Report

- ETHYL TERTIARY BUTYL ETHER ORAL ABSORPTION IN

MALE AND FEMALE RATS



### 5 9. Page 12 of 217, Section 3.7: Is Grubb’s test the

test that was used to exclude data points? It appears from the Results that the only data set that

was excluded from by the Grubb’s test was the data set from DF05, a low dose female. More discussion of this point is needed as it is very unclear as to why some points were included in the PK analysis and others were eliminated. 

### Disagree. Grub b’ s test was used to exclude one data point. The other points

were not excluded or eliminated from the analysis. They were not used by

WinNonlin to fit the terminal elimination phase of the PK analysis.

I believe that this reviewer does not have a clear understanding of how this PK analysis wa s conducted with WinNonlin. No additional data points were excluded from the analysis.

10 Page 14 of 217, Section 4.5: 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, since the absorption phase was not captured in the data

obtained. As ackn owledged by the authors in the final report, it is true that Cmax and Tmax could not be determined accurately. Was a pilot study done to determine the best sample time? It seems that with an indwelling cannula that samples could have

been taken as soon as 5 min after dosing. Cmax and Tmaxmight have been able to be determined with greater precision if samples included both the

rise and decline in concentration. 

### No pilot study was conducted.

11 Page 18 of 217, Table 1: What do “Timepoint ” and “0m” mean? It is the time that the body weight was determined? If so, is this column necessary since the time of body weight determination is described

in the footnote. 

### Deleted the column Page

12

12 Page 20 of 217, Table 3: How is “Dose error”

calcu lated? Can you add a footnote to the table to explain this term and how it was determined? 

### Added a footnote. 100* (( Actual dose

– target dose ) /target dose )

13 Page 22 of 217, Table 5: The choice of which data points to exclude from the PK analyses seems arbitrary. For example for the high dose females all

the 0.25 hr values were eliminated but for the males only 2 of the five were eliminated, and these values were not even the highest and lowest values for samples taken at this time point. 

### I disagree. Ag ain, these were not

eliminated or excluded . They were put into WinNolin and were used in the analysis,

bu t not used by WinNonlin to fit the

terminal e limination.

14 Page 36 of 217, Protocol Section 7: Male rats used in the study were less than the 200 g stated in the Protocol and females were much less than 200 g. 

### An approximate weight was indicated. No change.March 6, 2013 Audited Final Draft Report

- ETHYL TERTIARY BUTYL ETHER ORAL ABSORPTION IN

MALE AND FEMALE RATS



### 6 15 Page 49 of 217, Protocol Change 5: Clarification of flushing of cannulae to maintain patency should be more explicit in the final report. Was the cannula flushed with saline after each blood sample was

taken? This is not clear. The Amendment simply states following dosing. However, dosing is by gavage and does not involve a ca nnula. 

### Added clarification. The cannulas were left intact until the day the animals were dosed. They were replenished with heparinized saline after each blood sample was taken

16 Page 66 of 217, Method Validation Report: No dates or signature are on va lidation Report 

### Added signed signature page.

17 Page 80 of 217, Method Validation Report, Section 11.4: The final report notes that blood from ETBE exposed rats was stored at 4°C prior to analysis. However, the data on stability in blood suggests

that storage at room temperature for 8 hr yielded a value closer to the actual concentration, while storage at 4°C yielded values that were lower even though all values were within the acceptance

criteria. RTI notes that it was decided to store at 4°C to be pru dent, but it seems that room temperature yielded better results, so it is not clear what the lab meant by prudent. 

### Storage for a period longer than 8 hours was necessary accommodate all of the sample analysis.

QA Comments

1 ETBE levels reached maximal values at the first timepoint (0.25 hr) and declined rapidly thereafter, falling to less than that the limit of quantitation by 8 hr at the high dose and 2

- 4 hr at the low dose. 

### Changed ii

2 There were no significant protocol deviations (Appendix A) that would affect the integrity or quality of the study or the interpretation of the

results. 

### Changed iii

3 Copies of the approved protocol and protocol

amendments and deviations for these studies are included in Appendix A. 

### Changed 1

4 Identity and Purity; Details of the analysis conducted are contained in Appendix B C

. 

### Changed 2

5 Stability; Details of the analysis conducted are contained in Appendix B C

. 

### Changed 2

6 Blood samples (approximately 100 μl) were drawn from the jugular vein cannula (except those indicated in the protocol deviation list) into a heparinized 1 ml syringe, and the sample was immediately placed in a preweighed headspace

vial. 

### Changed 4

7 This included analysis of ETBE immediately after spiking in control blood, after storage at room temperature (8 hr), and or at approximately 4oC (24 hr). 

### Changed 4March 6, 2013 Audited Final Draft Report

- ETHYL TERTIARY BUTYL ETHER ORAL ABSORPTION IN

MALE AND FEMALE RATS



### 7 8 Calculations of sample data are described in Appendix F (?)G , and were reported with the DebraTM system. Is there an appendix G? I don’t see calculation descriptions. 

### Calculation descriptions have been added. 5

9 Blood concentration data for each dose group are reported in Tables 4

- 7 5

- 10

. 

### Changed 7

10 With gavage administration of 50 mg ETBE/kg,

ETBE reached a maximum in blood at the first timepoint (0.25 h), and declined rapidly, falling to <LOQ at 8 hr (Tables 4 and 5 7 ). 

### Changed 7

11 Similarly, with gavage administration of 10 mg ETBE/kg, ETBE reached 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 6 and 7 10 ). 

### Changed 7

12 No statistically significant. differences between

males and females were observed in the pharmacokinetic parameters measured. There were however differences between the high and the low dose groups that were similar in both males

and females. 

### Changed 8

13 Table 10 .0 Study Participants . Add Quality

Assurance Participants 

### Added 10

14 Deviation The protocol states (section 8.5.1) that: “The blood samples will be maintained at room temperature until analyzed.” They were maintained on ice until placed in the headspace autosampler for analysis. 

### Added A

- 22

15 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 temperature (8 hr), or at appro ximately 4oC (24 hr)." ETBE in

blood under all conditions tested was found to be

stable. 

### Added A

- 22

16 Question

- 1, 2, and 24 hour samples from DF05

were from cannulae while all others were from tail

vein? A

- 23March 6, 2013 Audited Final Draft Report

- ETHYL TERTIARY BUTYL ETHER ORAL ABSORPTION IN

MALE AND FEMALE RATS



### 8 17 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.
Question

– It is unclear why there were not sufficient extra animals for sampling at the earlier timepoints noted above. Are DF05 and BF03 already extra animals? There we re to be at least 1 extra animal for each group. 

### Perhaps a poorly worded deviation. In many instances we don’t use extra animals on study, because they aren’t required. It can depend on how well the cannulated animals from a specific vendor perform (how many loose patency). We base the number of extra animals on our experience with a particular vendor and animal strain. We are also constrained by our IACUC to avoid unnecessary animal use, so we take a middle ground. In this case, the number

of animal s that lost patency was higher than expected. DF05 was an extra animal. A

- 23

18 Should Table 3, Spiking Solution Aliquot volume be

0 and 0 for the two blanks? 

### Added 7 of 45

19 Tables 3, 6, 7. Indicate LOQ as required per Appendix F 

### Added Various