Document ID: EPA-HQ-OAR-2003-0065-0766
Agency: epa
Document Type: Supporting & Related Material
Title: 
Posted Date: 2015-10-02T04:00Z

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 continuously 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 gender (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
Section 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 range; 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 range on.
                                      11
                                      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 based 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 significant 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 below 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.  This information is included in Table 20.
                                      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.  
                                       2
                                       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 rats 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 similar to that found for females.

Male Rats
Time (hr)   Acetone μg/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 indicate that the behavior of the males and females was similar, with concentrations constant between 6 and 8 hours    
                                      11
                                       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 time 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 values for acetone in order to obtain estimates of the DIPE-derived 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 generating operator bias (cherry picking).  However, the data points used by the software may be different for each chemical in the same group of animals, or between groups of animals.  
                                       
                                       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 included 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 to the text.
                                      12
                                       7
Page 23 of 337, Discussion: The report notes the following "Comparison of the exposure of rats to 3600 ppm [2- 14C]DIPE/DIPE euthanized immediately following exposure (372 +- 8 mg/kg) with that of rats euthanized following 7 days of collection of excreta and exhaled breath (299 +- 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 time, 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 effects of the exposure duration and exposure concentration on the dose have been calculated.  Group B had an exposure duration of 6 hr, with a mean concentration of  3529 ppm, giving 21174 ppm.hr of exposure.  Group C had an exposure duration of 5.66 hr, with a mean concentration of 3334 ppm, giving 18870 ppm.hr of exposure.  Comparing the total radioactivity recovered normalized by exposure yields 0.0176 mg/ppm.h for Group B, and 0.0168 mg/ppm.h for Group C.  The normalized values for Group C is approximately 95.6% of that obtained for Group B.  This has been added to the Conclusion.
                                  14, 25, 35 
                                       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 equivalent 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 concentration 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 air 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 to 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. 

                                       6
                                      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 the 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 inhalation 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 concentration 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. 
                                       
                                      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 that 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 holding 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 modified 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 equilibrate 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 is 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 the later definitive studies.
                                       
                                      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 exposure. However, the same methodology for generating and sampling the exposure 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 sampling 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 differences 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 nose 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 exposure target concentration.  The differences in the sample times between Study A and Study B can be attributed to technician error.
                                       
                                      13
Page 263 of 337, Appendix E, Pre-Study Inhalation Report, Study C, Table 4: Similar to Study B the data for exposure concentration 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 exposure atmosphere recorded over 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 Study 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 concentration 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 running for 35 minutes when the first animal was loaded.) In Study C, there was a limited amount of [13]C6 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 be compromised. 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.
Added 
                                      Iv
                                       5
3.1.2 Cannot find supporting documentation for the purities reported here.
Added
                                       2
                                       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 ug/L, tertiary amyl methyl ether 0.1 ug/L, ethyl tertiary butyl ether 0.1 ug/L, diisopropyl ether 0.1 ug/L, and methyl tertiary butyl ether 0.1 ug/L).
                                       3
                                       7
3.4 Where is the stability reported?
It has been added in Appendix F
                                       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 ppm 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
                                      14
                                      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.
                                      14
                                      19
[b] Animals used in Group 3 and Group 4 ......"
Deleted
                                      17
                                      20
Table 2.  add footnote from Table 3
Added.
                                      18
                                      21
b Two animals with measurements less that the limit of quantitation.   
Changed
                                      23
                                      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.  
                                      26
                                      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 conducted 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 during 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 indicated 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 analysis, 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 calculation in Table 9, but excluded from the group mean and SD.  The data for recovery 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.   
                                       
                                      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 included in Appendix F
                                       
                                      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 open 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.