                                       
                                   COMMENTS
                               Action / Response
                                    Page #
                                       
   Dr. Schlesinger's Comments on the Draft Report of the Definitive Study


                                       1
p.16. Assignment of Animals to Groups. It would be very helpful if the investigators included a Table here indicating the Group Number related to the 4 objectives and exposure protocols noted on pages 13-14.  It is not easy to follow this from the text
Agreed.  It is difficult to follow.  We will revise the text to make this easier to follow, with in-text table similar to the protocol.  
16
                                       2
p. 18. Section 3.5. There appears to be a gap in group numbering per the description in text in that Groups 13 and 14 are not described here.
Groups 13 and 14 were removed from analysis.  We will revise the text to include truncated versions of Tables 1 and 2 from the protocol to clarify.  
18
                                       3
p. 25. From Table 11, the spleen had same amount as did the kidney in this case but this is not noted in text. 
Spleen has been added to the text
25
                                       4
p. 26. From Table 15, here again spleen and kidney are very similar yet this is not noted in text. Thus, there seems to be a pattern of high spleen levels in females. 
Spleen has been added to the text.  
26
                                       5
p. 26. It is noted here that is was suspected that samples were switched at collection but this could not be confirmed. Given that there is no definitive confirmation,  it is inappropriate to use these data at all and they should not be reported in the summary statistics.
While that could be a reasonable approach, it does not consider the manner in which the samples are collected and processed.  The samples are processed as they are collected, with each individual animal collected at intervals.  Because there are two trap samples collected and processed from one animal before the next animal samples are collected, the only logical explanation is that they were placed in the wrong vials for sampling, which are labeled, but differ in labelling by one number.   Rather than interfere with the mass balance and recovery, it is better to leave these in the statistics.
26
                                       6
P. 26. Adipose tissue is mentioned here related to results in Table 17 and 19, but it is not mentioned in relation to results shown in prior tables even though it is often high in those other groups as well.
Adipose is substantially higher than in other tissues in Tables 17 and 19.  It does not rise anywhere near these levels in other tables.  
26
                                       7
P. 29. It is noted that there was "little" difference between males and females in peak DIPE levels. This should be tested with some statistical test rather than using the term little. The same suggestion goes for AUC for DIPE. Furthermore, there should be some statistical analysis for the last sentence, namely that the Cmax and AUC values were higher in males than females. 
Because of the way the analysis was done for blood concentrations, and not all animals are sampled at the same time, it is not appropriate to analyze the PK data for individual animals which is what would be required for the evaluation of statistical significance.   
29
                                       8
Figures 6  -  11. There needs to be some notation as to whether the error bars are standard deviation or standard error. In addition, the labels for the Y axis are unintelligible. 
A notation of standard deviation has been added in the figure title.  The labels appear perfectly clear in our version.  The figure legend indicates that the Blood concentration is reported, and the y-axis scale indicates DIPE (ug/ml), acetone, or isopropanol.  No change to the figures.
74  -  79
                                       9
Section 13 in Appendix A outlines statistical testing that will be done, yet there is not indication in the body of the report that such testing was ever performed. There are no indications, as noted above, of the statistical significance of any differences noted. This needs to be addressed. 
Agreed.  The statistical analysis of the PK values can not be conducted, because of the way that the data was collected from different animals through the course of the study.  A protocol deviation has been prepared.  
A-26
                                      10
In [my] comments on the pilot study, it was noted, "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." However, in the current draft final report, the investigators indicate that "As was observed in the pilot study, comparison of the exposure of rats....euthanized immediately following exposure with that of rats euthanized following 1 day of collection...yielded values that were very similar. This suggested that there were no significant losses of radioactivity....during processing..." Firstly, these two comments need to be reconciled. Secondly, since no statistical evaluation was done, what is meant by "no significant losses?"
The second reviewer raised this as a concern.  We have revised the analysis in the pilot study, and improved the recovery somewhat.  Statistical analyses outlined in the protocol were conducted and added to the report.
30
                                       
                                       
                                       
                                       
                                       
                                       
                                       
                                       
                                       
                                       
                                       
                                       
                                       
                                       
                                       
                                       
                                       
                                       
                                       
                                       
                                       
                                       
                                       
                                       
                                       
                                       
                                       
                                       
                                       
                                       
                                       
                                       
                                       

	


                                       
     Dr. Bond  -  Summary Comments on Draft Report of the Definitive Study


                                       1
General Comments  -  See attached pdf file.
We appreciate Dr. Bonds comments and suggestions

                                       2
Recommendation regarding pentane contamination of radiolabeled solution - See attached pdf file

This is a good comment, and we will add a description of the pentane contamination to the final report, and include a column in Table 4 showing the potential exposure to pentane.  

                                       3
Recommendation regarding data presentation of metabolized dose - See attached pdf file
This is an interesting analysis, and could be added to the discussion.  However, for now we have not included it.

                                       



                                       
  Dr. Bond  -  Specific Comments on the Draft Report of the Definitive Study


                                       1
"This study evaluated the disposition of diisopropyl ether (DIPE) in male and Fischer 344 rats following nose only exposure and gavage administration, and pharmacokinetics following nose only exposure."

and female?
Added.  
i
                                       2
"Based on the recovered radioactivity at 24 hr, the estimated dose for male rats was 80.3  2.4 mg/kg, and female rats was 77.5  1.5 mg/kg."

These amounts are less than the amount measured at the end of exposure suggesting that all radioactivity was not recovered.
The statement in the report is accurate.  
i
                                       3
"Based on the recovered radioactivity at 24 hr, the estimated dose for male rats was 301.3  18 mg/kg, and female rats was 286  22 mg/kg."

Again, incomplete recovery.
The statement in the report is accurate.  
ii
                                       4
"The estimated dose recovered was 786 +- 9 mg/kg for males, and 480 +- 39 mg/kg for females."

This amount seems high relative to the dose recovered for the single exposure, and compared with the relative amounts recovered for females.
The statement in the report is accurate.  
ii
                                       5
"Based on the recovered radioactivity at 24 hr, the estimated dose for male rats was 543.9  11.2 mg/kg, and female rats was 401.8   59.2 mg/kg."

Why is there such a big difference between the dose recovered in males immediately after exposure (786mg/kg) and at 24 hr after exposure (543). On the surface this appears to be an anomaly, however, an alternate method for analyzing the data suggests that this difference is not unexpected.  See comments in review of overall report (another file).
The statement in the report is accurate.  
ii
                                       6
"A lesser amount was recovered (<1%) in feces, and approximately 5 % in the tissues and carcass at 1 day."

Recovery also about 80% for this gavage dose.
The statement in the report is accurate.  
ii
                                       7
"A lesser amount was recovered (<1%) in feces, and approximately 3 % in the tissues and carcass at 1 day."

Lower recovery (about 70%) at this higher gavage dose suggesting that not all volatiles were collected.
The statement in the report is accurate.  
ii
                                       8
"Male and female Fischer 344 Rats were used for the inhalation exposures to [2-[14]C]DIPE (Table 1), gavage administration of [2-[14]C]DIPE (Table 2), and inhalation exposure to unlabeled DIPE (Table 3).  Body weights and the number of animals"

This sentence needs to be completed.
The sentence has been deleted.
24
                                       9
"A larger amount (11.8-23.7 uCi) of radioactivity was retained following repeat exposure to 3600 ppm DIPE.  For the last radiolabeled exposure, the retained dose was higher in males than the single exposure, "  

What is the mg/kg for these groups?
The dose in mg/kg has been added. 
24
                                      10
"The estimated doses based on the total activity recovered at between the single exposure and 24 hr following the exposure were 80.3  2.4 (male, 600 ppm), 77.5  1.5 (female, 600 ppm), 301.1  18.4 (male, 3600 ppm), and 286.5  22.1 mg/kg (Table 7).  Following repeated exposures to 3600 ppm, followed by a single exposure to [2-[14]C]DIPE, the estimated doses based on recovered radioactivity were 543.9  11.2 (male) and  401.8  59.2 (female)  mg/kg."

Female, 3600 ppm?
Added to the text.
25
                                      11
"Table 4.  Specific Activity of Dose Formulations"

As indicated in written comments it would be useful to add the percent pentane contamination to this table.
Have added an extra column to Table 4.  

                                      12
  Time         Conc.      Pred.    Residual      AUC       AUMC      Weight
-------------------------------------------------------------------------------
83.29      397.0
      8.000 *      5.095      4.845     0.2497      87.78      431.1      1.000
      10.00 *      1.625      1.737    -0.1126      94.50      488.1      1.000
      16.00 *    0.08148    0.08013   0.001354      99.62      540.7      1.000
*) Starred values were included in the estimation of Lambda_z.

Why were only the last 3 data points used?
We fit the data automatically to a noncompartmental model, and let the computer fit the data.  It provides a best fit for the model, and selects the number of points that give the best fit.  It relieves the operator of questions of bias.   
F-15
                                      13
      6.500 *      51.28      55.39     -4.114      168.3      766.9      1.000
      7.000 *      43.17      42.41     0.7646      191.9      925.8      1.000
      7.330 *      38.68      35.55      3.130      205.4      1022.      1.000
      8.000 *      27.18      24.85      2.326      227.5      1190.      1.000
      10.00 *      7.378      8.536     -1.158      262.0      1481.      1.000
      16.00 *     0.3569     0.3459    0.01099      285.2      1720.      1.000
*) Starred values were included in the estimation of Lambda_z.

Here more than 3 data points were used.
Same explanation as above. We fit the data automatically to a noncompartmental model, and let the computer fit the data.  It provides a best fit for the model, and selects the number of points that give the best fit.  It relieves the operator of questions of bias.   
F-18
                                       
                                       


                                       
Dr. Bond's Comments on Test Chemical Analysis Report [2-14C]-Diisopropyl Ether


                                       1
["][1]H NMR spectra were acquired on [14]C DIPE prepared by adding 3 uL of [2-[14]C] DIPE (13426-4A) to 0.5 mL of CDCl3 on April 18, 2012 (exposure groups 5 and 6).  Spectra were acquired at 500 MHz with a sweep width of 5704.9 Hz, a pulse width of 11.3 usec." 

What does this lot number refer to?  Is this material diluted with unlabeled DIPE? Or is it neat material from the supplier?
This refers to a notebook (13426) and a page (4) and a sequential letter (A) used to reference materials that are used by an operator, so we can distinguish individual items by a unique reference, rather than the aliquot of DIPE prepared on a specific date.  This reference takes us directly to the notebook.  
4
                                       2
Following the exposure to the Groups 3 and 4, a sample of the remaining mixture used for the exposure was prepared for analysis (Figure 89).  
Corrected.
4
                                       3
"Our analysis of initial radiochemical purity indicated 97.3%."  

Radiochemical purity indicates that the 14C is associated with DIPE.
Is there a change requested?
5
                                       4
"However, analysis by NMR spectroscopy indicated that the material contained approximately 50% pentane." 

NMR indicates that the solution containing 14C-DIPE is about 50% pentane and 50% 14-C DIPE.
Again - is there a change requested?
5
                                       5
"Calculation of the degree of contamination resulting from the mass of pentane in the DIPE preparations was conducted prior to proceeding with studies using the [14]C DIPE." 

This is key to the integrity of the studies and should be presented in the main body of the final report and not restricted to an Appendix.  
We added text to the main body of the report. 
5
                                       6
"The dilution of the labeled DIPE with unlabeled DIPE results in a substantial dilution of the contaminants, with the resulting pentane amounting to approximately 0.5 % of the total DIPE/14C DIPE mixture used for the inhalation exposures conducted to date 3600 ppm DIPE.  We have verified that this is barely detectable by NMR spectroscopy as a contaminant in the final preparation used for the 3600 ppm exposure.  With our estimate of 0.5% of the material as pentane, the corresponding concentration of pentane during the 3600 ppm DIPE exposure would be approximately 25 ppm.  The OSHA PEL for pentane is 1000 ppm." 

[3600 ppm DIPE] It appears that the specific activity of the administered material (ratio of unlabeled DIPE to 14-C DIPE) is key to determining the percentage of contamination of the dosing solution by pentane. A table showing how the percentage of the pentane contamination changes as the test material is diluted with DIPE would be useful. This information is important for all of the studies -- inhalation at 600 and 3600 ppm and the gavage studies.

[exposure] This exposure would have the most dilution by pure unlabled DIPE.  What about the 600 ppm exposure and the gavage exposures.  What is the percent of contamination of pentane in these studies?
The amount of pentane in each of the dose groups has been added to Table 4.  The percentages are similar for the inhalation groups.  The gavage formulation had a higher percentage of pentane compared to DIPE, but with dilution by the vehicle ingredients (Cremophor and water), it was reduced substantially.    
5
                                       
                                       


                                       
Dr. Bond's comments on the Analysis of Oral Dose Formulations Diisopropyl Ether


                                       1
"From the calibration curve, the regression equation was used to calculate the concentration of DIPE in diluted samples and in samples."

Is something missing here?
Nothing missing.
4 of 18
                                       



                                       
      Quality Assurance Comments on Draft Report of the Definitive Study


                                       1
Radioactivity was eliminated primarily as exhaled volatiles (42 and 41 % of the recovered radioactivity, in males and females, respectively), and [14]CO2 (389  and 41%)
Changed
i
                                       2
In Groups 5 and 6, eight male and female Fischer 344 rats were exposed to 4 consecutive days of unlabeled DIPE at 3600 ppm,for 6 hr/day (actual concentration 3555 +- 55 ppm).  
Changed
ii
                                       3
At 1 day following the end of the last exsposure, the rats were euthanized and the radioactivity remaining in the tissues and carcass was determined by digestion and scintillation counting.  Radioactivity was eliminated primarily as exhaled volatiles (45 and 53 % of the recovered radioactivity in males and females, respectively), and [14]CO2 (34 and 26 %). Small amounts of radioactivity were recovered in urine (8.5 and 6.87 % of the recovered radioactivity).  
Changed
ii
                                       4
Small amounts of radioactivity were recovered in urine (28.5 and 2.2 % of the administered dose).  
Changed
ii
                                       5
Acetone levels in both male and female rats rose steadily during the exposure, and reaching a plateau following exposure until 8 hr following the initiation of exposure and then declined rapidly reaching preexposure levels by 16 hrs.

Indicates 7 hr in the results section  
Changed
iii
                                       6
Quality Assurance Statement - Needs to be completed.
Yes
iv
                                       7
There were no significant deviations (Appendix A) that would affect the integrity or quality of the study or the interpretation of the results.  Specimens, raw data, copies of the inhalation data, and the final report generated as a result of this study are archived at RTI International, until they are no longer needed for compliance (?).
Changed.  Deleted specimens, since we have indicated in the protocol that the DIPE will not be retained, and the specimens are not expected to afford reanalysis.  
v
                                       8
Three (?) unexposed rats (x ? male and X ? female) were housed in individual glass metabolism chambers for 1 day for collection of urine and feces for determination of radioactivity background.
2 male and 1 female.  Added
16
                                       9
The concentration of DIPE was monitored at the inlet to the tower, and at the outlet as described in the Inhalation Summary Report: Nose Only Exposures at RTI International ( Appendix EXX).
Changed
17
                                      10
The generation, exposure and monitoring systems are described in the `Inhalation Summary Report: Nose Only Exposures at RTI International'  Appendix EXX.  

Changed
18
                                      11
Table 7 and Table 8, and ranged from approximately 3.7 to 15.8 10.1uCi.
Changed
25
                                      12
Radioactivity was eliminated primarily as exhaled volatiles (Table 8 and Table 10) (42 and 41 % of the recovered radioactivity, in males and females, respectively), and [14]CO2 (389 and 41%) .
Correct as written  -  adding both the trap 1 and trap 2 means.  

25
                                      13
Small amounts of radioactivity were recovered in urine (8.5 and 6.87 % of the recovered radioactivity).  
Changed.
26
                                      14
As with the other? 3600 ppm exposure, highest concentrations were observed in liver, kidney, small intestine and lung.  
Changed to single 3600 ppm exposure.
26
                                      15
Small amounts of radioactivity were recovered in urine (82.5 and 2.2 % of the administered dose).  
Changed
27
                                      16
Additional tissues with similar levels were stomach, large intestine, spleen, and skin.
Changed
27
                                      17
The details of the inhalation exposure system are described in the report for the pilot study, (RTI-934, Metabolism and Pharmacokinetics of Diisopropyl Ether in Male and Female Rats: Pilot Study Appendix E, Inhalation Summary Report for Setup and Evaluation of Inhalation System at RTI International) and the details of the actual exposure are reported in Appendix EF, Inhalation Summary Report: Nose-Only Exposures at RTI International.  
Changed



28
                                      18
On exposure of rats to 3600 ppm DIPE, similar results were obtained.  DIPE accumulated rapidly during the 6 hours of exposure, and rapidly declined after the exposure (Table 31 and Error! Reference source not found.).  
Corrected to Figure 9.
29
                                      19
The peak concentrations of acetone achieved in male rats were approximately 50% higher than those in female rats.   Male rats reached preexposure levels by 24 hours.  Might be helpful to indicate that these were extrapolated values, not mentioned anywhere but in summary.
Added to the text.
29
                                      20
In these studies, approximately 12-13 11-12% of the radioactivity recovered was in the carcass and tissues in the male and female rats exposed to 600 ppm, and approximately 8 and 76 % of the radioactivity was recovered in the male and female rats, respectively, exposed to 3600 ppm.  With repeated exposures to 3600 ppm DIPE, approximately 110 and 121% of the recovered radioactivity was found in the tissues and carcass in male and female rats respectively.
Changed
30
                                      21
Quality Assurance Reviewer:  Add RTI Name

Deleted
32
                                      22
Table 2.  Body Weights[a] and Dose Administered to Male and Female Fischer 344 Rats in Oral Gavage Groups
Added footnote
34
                                      23
Table 10.  Add footnote from table 8

Added
44
                                      24
Table 16. Difference between 0 and 0.00*?
There are no instances of 0 in the table.  All values are reported to two decimal places and three sig figs.  *0.00 would represent a value that is below 0.005, and is less than 3x background.
50
                                      25
Table 20.  Percent of Administered Dose Recovered in Excreta and Tissues 24 h Following Gavage Administration of [[14]C]DIPE (50 mg/kg) to Male F344 Rats  (Group 7)
Added
54
                                      26
Table 22.  Percent of Administered Dose in Excreta and Tissues 24 h Following Gavage Administration of [[14]C]DIPE (50 mg/kg) to Female F344 Rats  (Group 8)
Added
56
                                      27
Table 24.  Percent of Radioactivity Recovered in Excreta and Tissues 24 h Following Gavage Administration of [[14]C]DIPE (200 mg/kg) to Male F344 Rats  (Group 9)
Added
58
                                      28
Table 26.  Percent of Radioactivity Recovered in Excreta and Tissues 24 h Following Oral Gavage Administration of [2-[14]C]DIPE (200 mg/kg) to Female F344 Rats (Group 10)
Added
60
                                      29
Table 33.  Extrapolated values?
A footnote has been added.
67
                                      30
Figure 11.  Blood Concentrations of Acetone in Male and Female Rats Exposed to 3600 ppm DIPE for 6 hr.
Male rat values here are not correct.  Values reported are well over 200 ug/ml

Correct.  This had been changed to the correct graph.
79
                                       
                                       


                                       
Quality Assurance Comments on the Analysis of Oral Dose Formulations Diisopropyl Ether


                                       1
Aliquots of each 100 ul dilution 100 ul were transferred into a 10-mL headspace vial, capped with a crimp seal cap, and .10 l of ISTD working solution was added through the septum using 10 l syringe.
Changed to Aliquots of each dilution (100 ul), made the second change
3 of 18
                                       2
Calibration curves were generated in Tablecurve.  
Corrected
4 of 18         
                                       



                                       
         211b Research Group Comments on Draft Definitive Study Report


                                       1
In Groups 1 and 2, [and 3 and 4] eight male and eight female Fischer 344 rats were exposed to [2-[14]C] DIPE in a nose-only inhalation system at a target concentration of 600 ppm for 6 hours (actual concentration 605  12 ppm).  
Corrected
i
                                       2
Non-compartmental pharmacokinetic analysis of DIPE indicted a terminal half life of approximately 1 hr at both exposure concentrations in male and female rats.  
I think that this is debatable as to whether this is a terminal half life.  It is the half life observed in this study.  
iii
                                       3
Oral administration of DIPE indicated extensive elimination of radioactivity by exhalation as CO2 and volatiles.  
Changed
iii
                                       4
The Environmental Protection Agency under the Fuel and Fuel Additives health effects testing program regulations codified at 40 CFR part 79,58(c) has indicated the need for data on the toxicity of oxygenated fuel additives, including diisopropyl ether (DIPE).   A component of the data required to evaluate the potential health risk(s) of DIPE is the investigation of the uptake, pharmacokinetics and metabolism of DIPE following inhalation (the expected primary routes of exposure) and oral dosing in rats.  Investigation of the kinetics of DIPE following single and repeated inhalation exposure will provide an assessment of the effects of exposure on the capability of DIPE to alter the capacity for its own metabolism by enzyme induction or inhibition.   

This opening paragraph needs to be more precise, particularly since as written there is no mention of oral studies which suddenly appear in 2.0.

Happy to change it.  Added oral studies
13
                                       5
Animals in Groups 1/2 and 3/4, were assigned Study IDs in order of eartag numbers from lowest to highest after animals that did not acclimate well to the nose only tubes prestudy were eliminated.   

Is this part of the lab's SOP? It could be a potential source of bias in subject selection, e.g. more agitated rats could be those with a higher metabolic rate. If it is SOP then disregard comment.
It is not an SOP  -  we had to do this to ensure IACUC approval for this study. 
16
                                       6
3.5.3	Single Exposures  -  Metabolism and Distribution
Eight male (Group 1) and eight female (Group 2) F344 rats  were exposed for approximately 6 hr to a target concentration of 600 ppm [14]C DIPE/DIPE via nose only inhalation.  Eight male (Group 3) and eight female (Group 4) F344 rats  were exposed for approximately 6 hr to 3600 ppm.
3.5.5	Repeat Exposures  -  Metabolism and Distribution
Eleven male (Group 5) and eleven female (Group 6) F344 rats  were exposed for approximately 6 hr/day to 3600 ppm unlabeled DIPE via nose only inhalation for 4 days.  On the fifth day, eight rats per sex were exposed for approximately 6 hr to 3600 ppm [14]C DIPE/DIPE.
3.6  	Oral Gavage Administration
Male (Group 7, N=4) and female (Group 8, N=4) rats were administered a single dose of [2-[14]C]DIPE/DIPE by gavage at 50 mg/kg.  Male (Group 9, N=4) and female (Group 10, N=4) rats  were administered a single dose of [2-[14]C]DIPE/DIPE by gavage at 200 mg/kg.




Changed








Changed






Changed
18
                                       7
Carcasses from Groups 11, 12, 15 and 16 were not analyzed
Changed
21

                                       8
Calculated doses (mg/kg) ranged from 96.8 mg/kg in females and 100 mg/kg in males at 600 ppm.

Averaged? These are not ranges. If means shouldn't they be +- SD?
These are means  -  changed the sentence
24
                                       9
The concentration of radioactivity in tissues at 24 hr are presented in Table 13 and Table 15.  As with the 600 ppm exposure, highest concentrations were observed in liver, kidney, small intestine and lung.  

The values in these tables are calculated concentrations of DIPE not amount of radioactivity.
Actually this is not the case.  The concentrations are calculated in DIPE equivalents and are radioactivity.  We don't know what the radioactivity is unless we separate it. So it is incorrect to refer to it as DIPE, but as DIPE equivalents.  We can change this to Concentration of Radioactivity (DIPE equivalents ug/g)
26
                                      10
For the purpose of reporting summary statistics, the value recorded for trap 2 has been switched with that recorded for trap 1.

Verify that this is noted in protocol deviations.
Deleted this wording and reported the data as is.  No deviation.  
26
                                      11
The concentrations of radioactivity in tissues at 24 hr are presented in Table 17 and Table 19.

Again, values are estimated DIPE concentrations.
See response to 9 above.
26

