March 6, 2013 Audited Final Draft Report - ETHYL TERTIARY BUTYL ETHER ORAL ABSORPTION IN 

MALE AND FEMALE RATS

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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 5th 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.

There are two completely differing 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 

parameters 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 parameters 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 Bond
March 6, 2013 Audited Final Draft Report - ETHYL TERTIARY BUTYL ETHER ORAL ABSORPTION IN 

MALE AND FEMALE RATS

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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 administered 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).

Disagree. 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 Because 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 administration. 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

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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 hours) 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 

complexity 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 

unclear 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 hour 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 points (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 

software 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

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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 for analysis and the complex, multiphasic nature of the time course after oral 

administration, no conclusions can be, or should 

be, drawn from the PK analysis.

I entirely disagree with the reviewer. The 

statement begins “Thus....” 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-compartmental

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 the 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 sample 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 missed, 

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 sampling 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 disagree with the assertion concerning 

the utility of the PK analysis. The analysis 

was not conducted with a onecompartment 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 noncompartmental 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

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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. Grubb’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 was 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 acknowledged 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” 

calculated? 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. Again, these were not 

eliminated or excluded. They were put into 

WinNolin and were used in the analysis, 

but not used by WinNonlin to fit the 

terminal elimination. 

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

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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 cannula.

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 validation 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 prudent, 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 thanthat 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 BC. 

Changed 2

5 Stability; Details of the analysis conducted are 

contained in Appendix BC. 

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 4
March 6, 2013 Audited Final Draft Report - ETHYL TERTIARY BUTYL ETHER ORAL ABSORPTION IN 

MALE AND FEMALE RATS

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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 4-75-10. 

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 57). 

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 710). 

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 Table10.0 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 approximately 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-23
March 6, 2013 Audited Final Draft Report - ETHYL TERTIARY BUTYL ETHER ORAL ABSORPTION IN 

MALE AND FEMALE RATS

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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 were 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 animals 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