March 24, 2013 Draft Report

- METABOLISM AND PHARMACOKINETICS OF DIISOPROPYL ETHER IN

MALE AND FEMALE RATS: PILOT STUDY



### 1 COMMENTS Action / Response Page

# Dr. Richard Schlesinger

1 I have reviewed the report and found that the original goals of the study have been met. The overall conclusions are supported by the reported results. Agree

2 There is no statistical evaluation in the Report. However, the investigators do compare various values between genders and at different times. It

would be best if there was some level of statistical significance associated with their value judgments. 

### Since this is a pilot study, to be repeated, we had avoided statistical comparisons.

3 Section 3.4. What was the time frame for sampling DIPE at the tower inlet and outlet, i.e., continuous, discontinuous, etc. ? 

### Continuous . Added “The

concentration of DIPE was monitored

continuou sly at the inlet to the tower,

and at the outlet.” 5

4 In Section 3.5, it is noted that for Study A, a total of 9 male and 9 female rats were exposed to labeled DIPE. However, the Protocol noted in Section 2.0 indicates that Study A involved use of non

- labeled DIPE and exposures were to be conducted on only 6

rats. This needs to be clarified. 

### This has been corrected. Rats were exposed to unlabeled DIPE in study A, with 8 animals exposed, and samples from 6 analyzed. Changed

9 to 8 in the text 5

5 Section 3.5.1. It is stated that the port to port variability was “within accepted limits.” Please define in the report body what these limits actually are. 

### Added criteria for acceptable performance. 6

6 Section 3.6.1. The blood sampling times in the first paragraph are unclear. It appears that 3 rats had blood drawn from 5 min to 6 hr after exposure began

and another 3 had blood drawn at 14 min to 18 hr after the exposures ended. What happened to the other 3 rats in each g ender (see #2 above) ? 

### In 4 above, the numbers have been clarified. Additional samples were collected to cover the possibility of loss of an animal. However, the duration of sample stability did not permit analysis of all of the samples

collected. 6

7 S ection 4.2 should be moved to the Methods section of the Report. 

### We see this as part of the data generated, and is appropriate for inclusion in the results. No change.

8 Section 4.3 and Table 4. Why is the exhaust concentration for Study C half that of the inlet for this protocol only, compared to A or B. 

### The flow rate of the tower was

reduced because of the limited

amount of 13 

### C label available

. 9 Section 4.4. The concentrations are in Figures 1

- 4, not just 1

- 3 as noted in the text. Furthermore, the first paragraph on pages 10

- 11 seems to have sentences that are repeated. Finally, why did the concentration

of acetone exceed the calibration curve ran ge; i.e., why did the investigators not anticipate this potential and adjust the range accordingly. 

### Corrected. Removed duplication.

Acetone concentrations exceeded our expectation, and we did not have any previous data to base the expected concentration r ange on. 11March 24, 2013 Draft Report

- METABOLISM AND PHARMACOKINETICS OF DIISOPROPYL ETHER IN

MALE AND FEMALE RATS: PILOT STUDY



### 2 10 Section 4.4. In the second paragraph, Figure 4 should be Figure 5, and Figure 5 should read Figure 6. 

### Corrected 12

11 Section 4.5. How was dose estimated? 

### It is b ased on the total amount of radioactivity recovered , divided by the specific activity. Added a footnote to Table 9 to clarify. 25

12 Section 5.0. The investigators noted that comparison of DIPE levels in rats immediately after exposure with levels in rats after 7 days of collection of excreta indicated that there were no signific ant losses of radioactivity from the sample types during processing. However, looking at the data there seems to be about a 20% loss between input and output, and to this reviewer that percentage can be perhaps considered as significant. 

### See discussion bel ow for comment

#7 from Dr. James Bond . We have 1) corrected a calculation error and 2)

corrected the dose calculated for the specific inhaled dose , and added to

the discussion. When corrected for the exposure concentration and duration a similar recovery per ppm.hr was found with Study B (3529 ppm x 6 hr = 21174 ppm.hr, 372.99 mg/21174 ppm.hr = 0.0173 mg/ppm.hr) and Study C (3334 ppm x 5.66 hr = 18870 ppm.hr, 317.89 mg/18870 ppm.hr = 0.16846 mg/ppm.hr). Since the recovery of Study C was 95.6% of Study B when corrected for exposure concentration and duration, we think that this recovery indicates no significant

losses occurred 14

Dr. James Bond

1 In general, the report is well

- written and thorough and the stated purpose of this study has been met. Thank you.

2 Page 2 of 337, Summary: There are several places in the Summary where there are typographical errors or missing text. These are noted as comments on the

Draft Final Report. 

### Corrected. 2March 24, 2013 Draft Report

- METABOLISM AND PHARMACOKINETICS OF DIISOPROPYL ETHER IN

MALE AND FEMALE RATS: PILOT STUDY



### 3 3 Page 20 of 337, Section 4.4: The final report notes that regarding DIPE concentrations in blood “There was little difference in blood levels at peak

concentration between males and females”. However, in Table 5 it appears that mean blood levels of DIPE in female rats appear to be consistently higher than

males throughout the exposure period. The errors

associated with these mean values for both males and females may make this difference not statistically significant. Has a statistical analysis been conducted

to demonstrate that the blood levels are/are not

different? 

### No statistical analysis was

conducted , since this was a pilot study, and no statistical analysis was indicated in the agreed protocol.

4 Page 21 of 337, Section 4.4: The report notes that

“Acetone levels in both male and female rat s rose steadily during the exposure, and in males continued

to rise following exposure until 8 hr following the initiation of exposure.” However, if one takes into

account the errors associated with the mean

concentrations of acetone in blood of male rats between 6 and 8 hours after the start of exposure (see relevant portion of Table 7 reproduced below), it appears that there is no difference among these values. These values appear to remain constant over this time period (i.e., 6 to 8 hours), a result sim ilar to

that found for females.

Male Rats Time (hr) A cetone μ g/mL Mean SD

6 288.42 76.855

6.25 298.9 19.328

6.5 319.38 15.137

7 312.47 22.028

7.33 315.15 19.22

8 301.87 16.539 

### The text has been changed to

indictate that th e behavior of the males and females was similar, with

concentrations constant between 6

and 8 hoursMarch 24, 2013 Draft Report

- METABOLISM AND PHARMACOKINETICS OF DIISOPROPYL ETHER IN

MALE AND FEMALE RATS: PILOT STUDY



### 4 5 Page 21 of 337, Section 4.4: The pharmacokinetic analysis of the decline in blood concentrations of DIPE, isopropanol, and acetone after the end of

exposure seems problematic. It is not clear why the same time points are not used for analysis for male and female rats for DIPE, isopropanol, and acetone.

For example, for acetone, time points from 7 to 24 hours post exposure are used for males, but tim e points from 6.25 to 24 hours post exposure are used for females, with no explanation as to why these earlier time points were not used for males also. Additionally, it is probably not appropriate to include

data on acetone concentration in blood from the 16 and 24 hour time points for females since these values are at background acetone levels. Inclusion of

these values tends to increase the half

- time by artificially elevating the values for acetone derived from DIPE. The fit of the predicted exponential equation to the data for blood acetone concentration in females in Figure 7 of the report shows that there is

not a good fit to these later time points. Background

should be subtracted from all va lues for acetone in

order to obtain estimates of the DIPE

- de rived acetone

which is critical at the lower acetone blood
concentrations. Since a radiolabel was not used in Study A it is not possible to differentiate endogenous

acetone from DIPE

- derived acetone. 

### For each analysis, WinNonlin was used for non

- compartmental analysis, and the analysis was conducted with the software choosing the best fit to the data. This avoids the perception of the operator choosing the fit that they like best, and generat ing operator bias (cherry picking)

. However, the data points used by the software may be different for each

animal.

6 Page 22 of 337, Section 4.6: The report notes that “The total recovery of radioactivity from all of the samples collected from rats in Group 3, Study C is presented in Table 9, and ranged from approximately 25 to 28 μCi.” How was this range determined? Table

9 shows a value for CM

- 04 that is 6.898. If this rat is

included in calculations then the mean value and range in Table 9 would also change. If this animal

was not i ncluded in the calculations it should be specifically noted. 

### There is a footnote in the table noting

that rat CM

- 04 was not included in the total analysis of samples, with the exhaled VOC traps reserved for possible later analysis. A note has

been added t o the text.March 24, 2013 Draft Report

- METABOLISM AND PHARMACOKINETICS OF DIISOPROPYL ETHER IN

MALE AND FEMALE RATS: PILOT STUDY



### 5 7 Page 23 of 337, Discussion: The report notes the following “Comparison of the exposure of rats to 3600 ppm [2

- 1 4C]DIPE/DIPE euthanized immediately following exposure (372 ± 8 mg/kg) with that of rats euthanized following 7 days of collection of excreta and exhaled breath (299 ± 7 mg/kg) yielded values

that were very similar. This suggested that there were no significant losses of radioactivity from the various sample types during processing of radioactivity.” However, the value of 299 is about 20% less than the value of 372 [calculated as follows: (372

- 299/372)*100]. This suggests that 20% of the radioactivity is unaccounted for when all routes of elimination and the material remaining in the carcass are quantitated. On the surface these data seem to suggest that about 20% of the radioactivity was lost during processing. However, there may be other factors that would result in a lower dose in the Group

C rats. Since the Group B and Group C rats were not exposed to DIPE at the same ti me, differences in the exposures may explain part of the difference in the body burdens of the two groups. The report should address the impact that a shorter exposure duration and a lower mean DIPE exposure concentration for Study C would have on the dose of DIPE that was absorbed by these rats compared with rats in Group B. These two factors can be, and should be, quantitated. 

### We did discover an error in the calculation of recovery for Group C,

which increased the dose for each

animal. The effect s of the exposure duration and exposure concentration

on the dose have been calculated. Group B had a n exposure duration of

6 hr , with a mean concentration of 3529 ppm, giving 21174 ppm. hr of

exposure

. Group C had a n exposure duration of 5.66 hr , with a mean concentration of 3334 ppm, giving

18870 ppm. hr of exposure. Comparing the total radioactivity

recovered norma lized by exposure yields 0.0176 m g/ppm. h for Group B, and 0.0168 mg/ppm.h for Group C. The normalized values for Group C is approxi mately 9 5.6 % of that obtained for Group B. This has been added to

the Conclusion.March 24, 2013 Draft Report

- METABOLISM AND PHARMACOKINETICS OF DIISOPROPYL ETHER IN

MALE AND FEMALE RATS: PILOT STUDY



### 6 8 Page 209 of 337, Appendix E, Pre

- Study Inhalation Report, Table 3: This study shows that the distribution of DIPE throughout the inhalation chamber is uniform. However, this table does not show that the
concentration AT THE BREATHING ZONE of the rats is equivalent to the target concentration or to the

DIPE concentration measured at the inlet. Demonstration that the concentration of the exposure atmosphere at the port is e quivalent to the

concentration at the inlet to the chamber is critical since it is the inlet concentration that is reported as
the chamber concentration. One cannot assume that the concentration at the breathing zone of the rat is equal to the concentratio n of DIPE at the inlet to the

chamber. The fact that these concentrations are about 48% of the target concentration of 3600 is problematic. The report notes that 200 ml/min of test atmosphere was sampled from each port and was

diluted with 200 ml/min of ai r before the diluted test atmosphere entered the MIRAN cell. This would suggest that the concentration of the test atmosphere at the port was twice the concentration listed in Table 3 unless some adjustment, which is not described in the report, was used t o account for this dilution. The quantitative relationship between the port concentrations and the inlet concentration, and the consequences of the port/inlet concentrations not being equal should be addressed. 

### The reviewer correctly notes below

that the reduction in concentrations

measured and concentrations at the

inlet result from additional air added to make up the flow rate into the Miran detector from the ports. The port measurements were made to

establish port to port variability and were diluted to enable more rapid sampling at a number of ports. This has been clarified in the final report. The sampling was conducted to show port to port variability, and

9. Page 210 of 337, Appendix E, Pre

- Study Inhalation Report, Table 4: The daily mean concentration for the Inlet in Table 4 suggests that the concentration at the breathing zone (1720 ppm for the 17 open ports taken

from Table 3) is from 56 to 50% of the inlet concentration depending on when that concentration was measured (averaged over the whole exposure or during the last 2.5 hours). This comparison raises several important issues. First, why is the concentration at the breathing zone so much lower

than the inlet concentration? Second, why is th e concentration measured at the Inlet so much higher during the last 2.5 or 3.5 hours of the exposure? Third, why is the exhaust concentration higher than the inlet concentration? These are important issues,

critical to the demonstration that the inhalatio n exposure system can deliver the target concentration to the breathing zone of the rats, and should be

addressed. 

### As noted in 8 above, the

concentrations were determined after dilution with an equal volume of air, as noted in the report. The

concentratio n is not lower in the breathing zone. The concentration

measured at the inlet was obtained by passing 200 mL/min into the inlet Miran, whereas the concentration at

the outlet was measured with the

entire effluent from the tower. This was modified during the definitive

studies so that the entire flow onto the tower passed through the flow

cell.March 24, 2013 Draft Report

- METABOLISM AND PHARMACOKINETICS OF DIISOPROPYL ETHER IN

MALE AND FEMALE RATS: PILOT STUDY



### 7 10 Page 21 of 337, Appendix E, Pre

- Study Inhalation Report, Study A, Table 5a: The data in this table suggests that it took 4.4 hours for the exposure
system to reach a stable concentration. This is problematic if this inhalation methodology is used in the animal studies because in Study A, for example, blood samples are being taken from rats starting at 5 minutes into the exposure. Table 5a would then suggest th at in Study A the lower blood levels of the

rats during the early times in the exposure could be a combination of short exposure duration and lower exposure concentration. It would be preferable to start the exposure of the animals (i.e., plug the tubes

ho lding the animals into the ports) AFTER the

chamber had reach a constant concentration. 

### If you examine the data in Table 4 for Study A

, the inlet concentration was approximately 99% of the maximum concentration by 75 minutes. The inlet sampling was modifi ed for the

later definitive studies.

11 Page 231 of 337, Appendix F, Inhalation Reports, Study A, Table 4: The data in this table is problematic for a number of reasons. First, it does not appear that DIPE concentrations reached steady

- state until 105 minutes into the exposure. Does this mean that the rats were initially exposed to a lower concentration of DIPE for the first 100 minutes? This is problematic because blood samples were taken from the rats as soon as 5 minutes into the exposure where they would be exposed to concentrations of DIPE that were only 82% of the concentrations from 105 minutes until 405 minutes. However, the exhaust concentration appears to be stable from the first sampling point (15 min) until the end of the exposure, suggesting that it is the sampling methodology, not the generation of the exposure atmosphere that is an issue. It appears that the problem is the time that it takes to equilibrate the MIRAN (>60 min) that is analyzing the air sample taken from the inlet. Time to

eq uilibrate is not a problem for analysis of the exhaust

since 100% of the exhaust enters the MIRAN that is analyzing that concentration. It is likely that the “real” exposure concentration in this study is approximately 3700 ppm, and the exposure atmosphere samples taken up to and including 75 minutes are not representative of the chamber concentration, but rather representative of the time it takes to equilibrate

the MIRAN. Inclusion of these low values in the

calculation of the mean chamber concentration i s misleading an artificially lowers the average for the exposure concentration. 

### If you examine the data in Table 4 for Study A, the inlet concentration was approximately 99% of the maximum concentration by 75 minutes. The inlet sampling was modified for t he

later definitive studies.March 24, 2013 Draft Report

- METABOLISM AND PHARMACOKINETICS OF DIISOPROPYL ETHER IN

MALE AND FEMALE RATS: PILOT STUDY



### 8 12 Page 247 of 337, Appendix E, Pre

- Study Inhalation Report, Study B, Table 4: The data for the exposure concentration at the inlet indicate that for Study B the exposure concentrations had reached steady

- state by the first sample time, 10 minutes into the e xposure

. However, the same methodology for generating and sampling the ex posure atmosphere that was used in Study B was used in Study A where it took approximately 100 minutes to reach steady

- state. In fact, the exact same Miran IR spectrometer (Serial number 4121) was used in both studies. Thus, with the same methodology and s ampling system, it is not clear how the laboratory was able to generate analytical data to suggest that steady

- state
concentrations were achieved in 10 minutes in one study, but not until 100 minutes in the other study. Some explanation as to the differenc es between the two exposures in sampling or analysis is required. 

### There were no differences in the exposure sampling or analysis between Study A and Study B. In Study B, the raw data indicates that the quantity of DIPE displaced once the syringe was loaded was greater

than 0.18 mL. This caused the

concentration on the no se only tower to go higher than the target concentration of 3600 ppm. The syringe pump was turned off and the

excess test material was allowed to evaporate until the concentration

value at the inlet Miran and the outlet

Miran were within 10% of the

exposur e target concentration. The differences in the sample times between Study A and Study B can be

attributed to technician error.March 24, 2013 Draft Report

- METABOLISM AND PHARMACOKINETICS OF DIISOPROPYL ETHER IN

MALE AND FEMALE RATS: PILOT STUDY



### 9 13 Page 263 of 337, Appendix E, Pre

- Study Inhalation Report, Study C, Table 4: Similar to Study B the data for exposure concen tration at the inlet indicate that for Study C high concentrations of exposure atmosphere were attained by the first sampling point, in this case

20 minutes . In fact, this 20 minute sample is the highest concentration of the ex posure atmosphere

recorded ov er the entire 335 minutes of sampling. Again, the same methodology for generating and sampling the exposure atmosphere was used in Study C as was used in Study A and again the exact same Miran IR spectrometer (Serial number 4121) was used. Thus, with the same methodology and sampling system, it is not clear how the laboratory was able to generate analytical data to suggest that the maximum exposure concentration was achieved by 20 minutes into the exposure. Some explanation

as to the differences between St udy A and Study C in sampling or analysis is required. 

### There were no differences in the exposure sampling or analysis between Study A and Study C. At

the time the first animal was loaded

the inlet concentration was within 20% of the exposure target

concen tration of 3600 ppm and the

raw data indicates the concentration value measure by the inlet Miran was still increasing and had not reached a steady

- state. (NOTE: The amount of

time needed to fill the inlet Miran cell

is 29 minutes

– the system had been

run ning for 35 minutes when the first animal was loaded.) In Study C, there

was a limited amount of 13 

### C 6 

### test
material available. Calculations on the exposure day indicated that the amount of test material remaining in the syringe had reached a critical point and the decision was made to start loading animals onto the nose only tower, otherwise the entire exposure would compromized . Study records indicate the actual exposure length was ca. five hours and forty minutes. A Protocol Deviation Report

documents the Deviation, Reason, Study Director Recommendations and indicates that the Sponsor was

contacted.

QA Comments

1 Summary: Second paragraph ends in mid

- sentence. 

### Missing text has been added 1

2 Summary: Last paragraph says “...for inhalation exposure to DIPE and rr achieved.” Please correct. 

### Corrected 2

3 Need to complete QA statement 

### Will complete when finished Iii

4 Need to add API signature block for GLP statement. Iv

5 3.1.2 Cannot find supporting documentation for the purities reported here. 

### Added 2March 24, 2013 Draft Report

- METABOLISM AND PHARMACOKINETICS OF DIISOPROPYL ETHER IN

MALE AND FEMALE RATS: PILOT STUDY



### 10 6 3.3 Diet. Protocol indicates that waters samples should have also been checked for the presence of TBA. Where are the results of the water sample

tests? 

### All were below the reported limit of

detection and text has been added : All of the analytes were below the reporting limit for the method in the samples obtained before and after the study (tertiary butanol 1 μg/L, tertiary amyl methyl ether 0.1 μg/L, ethyl tertiary butyl ether 0.1 μg/L, diisopropyl ether 0.1 μg/L, and methyl tertiary butyl ether 0.1 μg/L). 3

7 3.4 Where is the stability reported? 

### It has been added 5

8 4.1 99.71 ± 0.01% Where is this purity reported? Cannot verify. 

### Reported in revised Appendix B,

Table 1. 9

9 4.1 Cannot find results of NMR spectroscopy analysis. 

### Added Appendix containing the data 10

10 4.3 Appendix F indicates the name of the report is, Inhalation Summary Report: DIPE , 14C

- DIPE/DIPE,

and 14C

- DIPE/13C

- DIPE

. 

### Changed 10

11 4.4 Please indicate that “In the male rats , blood

concentrations of isopropanol were below LOQ...” 

### Added 10

12 4.4 top of page 11. Please indicate that “In the female rats, isopropanol concentrations were below
LOQ....” 

### Added 11

13 4.4 last paragraph. Please recheck all the Figures indicated. It appears that they off by 1 (i.e. 5 should be 6) 

### Changed 11

14 4.5 “... approximately 33 to 35 uCi.” 

### Changed 12

15 4.6 3 rd paragraph. “...at 8 and 24 hours, respectively.

Less than or equal to 0.2% of the radioactivity...” 

### Changed 12

16 5.0 “...3600 p p m DIPE.” 

### Changed 13

17 5.0 3 rd paragraph. Please recheck the percent 14CO2 recovery and the percent in the carcass

recovered. 

### Corrected . 20.2 % for CO2, and

2.6% for carcass 1 4

18 5.0 Last paragraph. “... 10 hours after initiation of exposure.” “... radioactivity in the exhaled breath and

as 14CO2.” 

### Added “initiation of ” exposure. Actually meant both the exhaled

breath volatiles and CO2. Added

clarification. 1 4

19 b Animals used in Group 3 and Group 4 ......” 

### Deleted 17

20 Table 2. add footnote from Table 3 

### Added. 1 8

21 b Two animals with measurements less that the limit of quantitation. 

### Changed 2 3

22 Table 10. The results for the second two animals in Group 2 Study B/Exp. Urine Sample 2 should be N/A 

### Added NS. 2 6March 24, 2013 Draft Report

- METABOLISM AND PHARMACOKINETICS OF DIISOPROPYL ETHER IN

MALE AND FEMALE RATS: PILOT STUDY



### 11 23 Table 11. Is N.S. not sampled? Where are the results of the polyethylene bag that held the animals during transfer? 

### N.S. is no sample. The animals were transferred inside the nose only tube into the Tedlar bag for euthanasia. Any radioactivity that was in the nose only tube or spilled out into the Tedlar bag was included in the exposure urine, exposure feces, or nose only

tube rinse. 26

24 Table 18. Where does data from “0” time point come

from? 

### This comes from the materials

collected as the animals were placed in metabolism cages. 33

25 The protocol indicates that the exposure atmosphere generation design was to be developed at CIIT.

It further indicates that the design technology will be reproduced at RTI and that verification studies will be

cond ucted without animals to assure that exposure atmospheres can be produced in the setup at RTI. It implies that the distribution studies will be done when

the verification studies are done, at RTI. The distribution study; however, was performed at CIIT

d uring the development and testing of the system. Work performed at CIIT is indicated to not be incompliance with GLP regulations. The distribution study is a GLP requirement and should have been conducted in compliance with GLPs. This will need to

be in dicated in the GLP Compliance Statement of both the main study and the Inhalation report. 

### This will be indicated in the compliance report for the main study.

26 Appendix E and F are signed as final. If any changes are made to these, they should be done as amendments to the report. 

### Noted.

27 Table 9 indicates that the data reported does not include exhaled breath traps, which were saved for other possible anal ysis, yet in the raw data there appear to be data for exhaled CO2 traps, urine, feces,

and carcass. Should these values be reported? 

### Traps from one animal were reserved. The other samples were analyzed for that animal. They were

included in the calculat ion in Table 9, but excluded from the group mean

and SD. The data for recover y expressed as a percentage of the total recovered would give an inappropriate comparison with the other animals, so the data has not

been included in Tables 11

– 18.March 24, 2013 Draft Report

- METABOLISM AND PHARMACOKINETICS OF DIISOPROPYL ETHER IN

MALE AND FEMALE RATS: PILOT STUDY



### 12 28 Table 18: This table is to include recovery of radioactivity in excreta and carcass, yet there is a 0 hour timepoint. Wasn't only blood was taken at 0

hours? Where could this data come from? 

### Table 18 includes the samples collected at each timepoint. 0 h included the nose only tube wash, and any urine or feces collected prior to the animals being placed in the metabolism cage.

29 Appendix E, Table 3: What are the units of measure for the reported values? Since the test atmosphere

was further diluted to reduce sampling time, 3600 ppm wasn't really the Target concentration. 

### The variability was what was being

measured here.

30 Appendix B, Page B

- 3, 5th paragraph: Is the word 'signal' correct, or is 'singlet' correct? 

### They are both correct. However

“ singal ” has been corrected.

31 Appendix B, Page B

- 5, Peroxide Check: Last sentence might be better as 'A white color indicated the peroxide value was 0 mg/L, through the storage period.' 

### No change

32 Appendix B, Page B

- 5, Conclusion, last sentence: ....

FID was 99.63 %.... 

### Corrected.

33 Appendix B, Page B

- 11: Was a stability test done after the study? If so, it should be included here. 

### Yes. It has been added.

34 Appendix C, Page C

- 4, Conclusion: The purity of the

test substance was 97.23% (?) by HPLC... 

### This has been corrected

35 Appendix F, Study A, page 10 of 16 (F

- 11), Nose Only Exposure System Distribution: The distribution study was performed at CIIT, not RTI. 

### The title of the report in Appendix E is

as shown.

36 The same is true for Study B, page 10 of 16 (F

- 27), and Study C, page 10 of 16 (F

- 43). 

### The title of the report in Appendix E is

as shown.

37 Appendix F, Studies B and C, Generation and Chamber Concentration, Pages 10 of 16 (F

- 27 and F

43): In the second paragraphs of the 'Generation and

Chamber Concentration' sections indicate the actual

DIPE that was used. For Study B, it was 14C

DIPE/DIPE and for Study C, 14C

- DIPE/13C

- DIPE. 

### The test chemicals used is clearly indicated throughout the text with this one exception. No change.

38 Appendix F: Throughout this appendix it is indicated that the stability of the test atmosphere was checked twice during the exposure by RTI (Page 7 of 16

in Study A, B, and C reports). Where are these analyses reported? 

### These have been includedMarch 24, 2013 Draft Report

- METABOLISM AND PHARMACOKINETICS OF DIISOPROPYL ETHER IN

MALE AND FEMALE RATS: PILOT STUDY



### 13 39 Appendix F: For Study C, the exhaust concentrations appear to be about half of the inlet concentrations. In the preliminary work done with 7 ports ope n and all of the other studies, the inlet and exhaust concentrations appear more similar. Any reason for this difference

in Study C? Was the exhaust Miran sample further

diluted for some

reason? 

### In the preliminary work, there are no animals to take up the DIPE. This is a reflection of the uptake of DIPE.

40 Appendix G: Page numbers for this appendix appear

as H

- #s. 

### Corrected

41 Appendix H, Raw Data Tables, Table 7: The header

on these tables indicates ETBE not DIPE. Please verify that the data reported are actually from the DIPE study. 

### Corrected

42 Appendix H, Raw Data Tables, Table 2, Blood Sample weights for Animal AM

- 06: Data for this animal appears twice. Could there be a mix

- up or just

clerical error? 

### Corrected

43 Appendix J: The note on the cover page indicates that this appendix will be updated with 'final version'. What will change? 

### The analysis was redone with a

validated version of WinNonlin following completion of review.