### 211(b) Toxicology Research Group DIPE One-Generation Reproduction Toxicity Study Report Reviewer Checklist
PP CS LR checklist reviewers; RG response to comment;

1 7/27/2010 

### Lab response to comments
Comments Action/Response Page
EPA In general, the NHEERL reviewers concluded that the studies were conducted in accordance with the testing guidelines, once changes detailed in the API peer review response are made. No comment

Additional comment is requested on the following points: Page 38 reports several statistically significant changes in reproductive organ weights (testis, epididymis and seminal vesicle/coagulating gland) at high dose exposures. The 17% increase in seminal vesicle/coagulating gland weight at 20,000 mg/m3 was dismissed as being spurious, due to the absence of histopathological changes, and the reproductive NOEL was set at 20,000 mg/m3. However, since an increase in seminal vesicle we ight is routinely used as a marker for androgenic activity, we respectfully submit that the reproductive NOEL be designated as 10,000 mg/m3. THE RG does not consider the increase in seminal vesicle/coagulating gland weight to be sufficient effect to alter the reproductive NOAEL from 20,000mg/m3. In addition to the absence of histopathologic changes, the parameters for sperm number, motility and morphology, and other reproductive parameters were unaffected by treatment. However, the sentence in Results, p. 38. will be revised to reflect that, rather than “spurious”, the weight change was not considered to be of definitive toxicological significance. Sentence clarified as su gg ested above. P. 40 √

The NOAEL of 20K is also consistent with the interpretations of our external peer reviewers.



### Peer Reviewer – Schlesinger
The study was conducted in a scientifically sound manner and followed the appropriate protocols. There were no significant deviations from these protocols that would have affected the outcome of the study. However, there appear to be an inordinate number of technician errors reported. In any case, the conclusions as to NOELS are supported by the data. Nearly all of the protocol deviations were related to isolated errors in data collection. None significantly impacted the overall results or conclusions of the study. While the absolute number of protocol deviations may seem excessive, a One Generation Reproduction study is a considerable undertaking and involves a large number of animals. The fact that these protocol deviations were noted and documented reflects positively on the Quality Assurance practices that have been put in place by API, and at the laboratory. Nonetheless, the RG has brought this issue to the attention of the laborator y

. The following issues should be addressed in the final version of the report:

### 211(b) Toxicology Research Group DIPE One-Generation Reproduction Toxicity Study Report Reviewer Checklist
PP CS LR checklist reviewers; RG response to comment;

2 7/27/2010 

### Comments Action/Response Page Lab response to comments

p. 28. Statistical Analysis: 2.16.1. It is noted that the mean values of all exposure groups were compared to the mean value for the c ontrol group at each time interval. It is not clear whether a one-way ANOVA was performed at each time interval so that multiple ANOVAs were thus used. Is there any reason why two way ANOVA was not used? This would have allowed evaluation of exposure groups at all times for each endpoint. This could have been followed by the appropriate multiple comparison test. The computerized statistical testing package employed at the laboratory is designed to perform one-way ANOVA at each time interval and is used for a range of multiple dose studies. The text will be clarified. Text in 2.15.1 clarified for 1-way test. I did not see a revision__same text but wording is same or similar to that in accepted reports

Why were tests conducted at both the 1% and 5% level of significance. The tests and level to be used should be determined a priori before the study begins and only one such level of significance should be used. These levels of significance were preselected since they were inherent in the laboratory’s computer statistical package. Comparison at two levels of significance has been common practice in this laboratory. It is noted that, "Dose groups were eliminated from statistical analysis if their standard deviation was 0 and/or N...in the group was less than or equal to two." Some indication should be noted as to how many such cases were encountered in the study. This is a standard statement for the statistical methods section. In this study, Table 1 Summary of Survival and Pregnancy provides examples of where this statement is relevant. These examples include data for Total females died/sacrificed; Nonpregnant animals, and No. of females cohabitated without evidence of pregnancy. These events are also observed in macro/micropathology incidence data. No statistical analyses are applied. p.32. Chamber Monitoring. The nominal and analytical concentrations are really quite close and I do not see a need to provide any explanation as to the differences. The explanation provided leads one to question why the calibrations of the chamber airflows and IR monitor were not done more carefully. Dr. Schesinger is correct. The sentences addressing reasons for possible differences between analytical and nominal values do not apply here and will be deleted from the Results and Summary sections. The differences were less than 3% and therefore considered carefully calibrated. p.33 reasons deleted √

The investigators need to explain the source of the particles in the exposure atmospheres. Was the incoming chamber air filtered appropriately? Any particles could act as adsorption sites for vapors in the exposure atmosphere. The mean particle concentrations were comparable between the control and treatment group chambers. The primary purpose of making these measurements in a study of this nature (high vapor concentrations) is to make certain that vapor condensation is not occurring and to preclude the possibility of aerosol formation. The results provided in the report confirm that gasoline + DIPE exposure was to vapors and not aerosols. √

### 211(b) Toxicology Research Group DIPE One-Generation Reproduction Toxicity Study Report Reviewer Checklist
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3 7/27/2010 

### Comments Action/Response Page Lab response to comments

Some particles are invariably present in chamber atmospheres, representing background levels. In fact, careful analysis of the data in the table on page 33 indicates that the particle concentrations in all of the chambers including controls ranged from 6- 8micrograms/cubic meter of air. For comparison, it has been reported in EPA's PM 10 Criteria document that annual background PM 10 levels in eastern cities range from 5 to 11 micrograms /cubic meter of air. Thus, the RG believes that the particles in the chambers reflect ambient background
values. p. 34. Maternal Body Weight. Effects seemed to occur at the mid and high exposure levels, so there does appear to be some consis tent exposure concentrationrelated effect. There were no statistically significant changes in maternal body wt during gestation (absolute [Table 7] or wt gain [Table 8]) and no dose-responsive effects during lactation (Tables 9, 10) where values were comparable to or slightly higher than controls. The slightly greater weight gain during gestation, well within normal variation, is most likely due to the slightly larger litter sizes in the exposed groups. The variations in gain and loss patterns among the groups during lactation suggest that these weight changes are natural fluctuations in lactating animals and not linked specifically to exposure. The RG agrees with the study director’s assessment and will discuss addition of more detail on p. 34 to justify the conclusion. Above discussion has been integrated
into R/D. p. 36

√ Okay to me.

p. 35. Maternal Feed Consumption. It is not clear why the disclaimer used here is that effects were not considered to be adverse while in other sections, thedisclaimer is that effects were not in a treatmentrelated pattern. According to Table 13, all treated groups had slightly higher feed consumption than controls with statistically significant increases seen only in the mid dose group during lactation days 1-4 and 1-28. The observations were not considered “adverse responses” because modestly increased feed consumption (as opposed to decreased feed consumption) especially in the absence of a dose

res p onse p attern is not identified as a p.36-37

√ Higher food consumption during LD 1-4 would be expected for larger litter sizes – no changes needed.

### 211(b) Toxicology Research Group DIPE One-Generation Reproduction Toxicity Study Report Reviewer Checklist
PP CS LR checklist reviewers; RG response to comment;

4 7/27/2010 

### Comments Action/Response Page Lab response to comments

potentially toxic effect. The RG will discuss revision of this sentence with the study director to clarify this conclusion. Above discussion has been integrated into R/D. p. 36. Parturition. The last sentence concerning statistical differences is unclear. The statistical differences questioned by the reviewer as" unclear" relate to “better-than-control pup survival during days 1-4”.
This section has been clarified. p.38

√ Okay to me.

p. 37. Body Weight. The statement concerning litter size effects on statistical analysis is not clear. Table 18 appears to show a real effect. From Table 16, it is clear that the Control group litters contained fewer pups by d. 4 of lactation than any treatment group: 11.1 in Controls, compared to 12.8, 13.4, and 12.5 pups/litter in low, mid and high dose groups respectively. The more pups competing for the dam’s milk supply, the lower the average pup weight/litter resulting in the statistically significant lower average pup wt in the 10,000mg/m 3 group and similar lower wt in the low and high dose groups compared to controls. When litter sizes were standardized on d. 4 (all litters with >10 pups were reduced to 10), all subsequent weights were comparable among treated groups and controls. An analysis of covariance could verify the relationship between litter size and average pup wt for these data. However, the RG agrees with the study director’s interpretation but will discuss including more detail in the explanation. This section reviewed again and considered OK as is. Okay to me.

p. 37. Terminal Estrous and Macroscopic Postmortem

. Data in Table 19 seem to show an exposure concentration-related effect in lung, discolored foci Thus, this may be treatment related. Dr. Schlesinger is correct that the number of rats with discolored foci in the lungs was observed to increase in an exposure-related manner in both sexes at necropsy of parental animals. As reported in the text, no histopathological effects such as alveolar macrophage aggregates consistent with discolored foci were seen and no changes in lung wt occurred. Therefore, the increased frequency of these background lesions is likely a marker of substance exposure but is not considered to be of adverse biological significance. The RG will discuss revisions of the text with the study director to better reflect this
assessment. This section reviewed a g ain and p.39 Ok to me.

### 211(b) Toxicology Research Group DIPE One-Generation Reproduction Toxicity Study Report Reviewer Checklist
PP CS LR checklist reviewers; RG response to comment;

5 7/27/2010 

### Comments Action/Response Page Lab response to comments

considered OK as is. p. 38. Organ Weights. Does the lack of correlation with histopathology indicate no adverse effect in spite of potential enzyme induction induced by exposure? Increased liver weights (absolute and relative) in the absence of corresponding histopathology findings are often seen to reflect a generalized adaptive metabolic response to exposure in studies of a variety of chemicals, rather than a unique adverse effect of an individual test material. The effect plays a significant role in establishing the NOAEL (see Conclusions, p. 39). The text on p. 38 of the Results section accurately describes the events in the liver. No changes in text have been made. Ok to me.



### Peer Reviewer – Goldsworthy
The study was scientifically sound and was properly conducted. Exposures were properly conducted and adequate to test the potential reproductive toxicity of Gasoline DIPE Vapor Condensate. None of the protocol deviations noted would be expected to adversely affect study results or data integrity. Protocol deviations listed on page 31 did not note the temperature or humidity excursions outside the specified range (page 19). Excursions outside the acceptable temperature and humidity range have also been cited by a RG internal reviewer. We do not consider these temperature and humidity excursions to be protocol deviations. p.19 no change to

text Response from SD elsewhere is that these excursions were consistent

across groups. No effect of treatment was noted on survival, clinical observations, body weights, feed consumption, estrus cycles, mating, fertility or gestation indices, or on other reproductive parameters up to parturition. No treatment differences were observed with pregnancy rates, duration of gestation and percent of females completing delivery. No differences between treated and control groups were noted in all parturition parameters, including total number of pups delivered, viability and lactation indices, the number of implantation sites per litter, the pup sex ratio, and the number of live pups per litter. Additionally, sperm count, motility, morphology and caudal epididymis weight were not altered by treatment. No comment

There were no exposure-related differences in macroscopic postmortem assessments. Increased kidney weights were observed in male rats (parental generation) at the two highest exposure levels; this is consistent with the expected induction of hydrocarbon nephropathy. Microscopic evaluation was not performed to confirm this observation . Hydrocarbon nephropathy is generally not c onsidered to be relevant for human risk assessment. There were statistical Dr. Goldworthy’s comment is correct. Although the Material & Methods section (Table 1, p. 25) stated that kidneys would be examined histologically from male rats in the control and high dose group, the data were not included in the draft report. The data will be added to the revised draft and results and discussion will be clarified. There were no findings of h y drocarbon ne p hro p ath y in the males OK I recommend the following change to the text in Section 3.4.2: From: “However, these same findings were not

seen microscopically with these test

### 211(b) Toxicology Research Group DIPE One-Generation Reproduction Toxicity Study Report Reviewer Checklist
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6 7/27/2010 

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differences noted in increased liver weights in male rats from the mid and high dose treatment groups. There were no exposure-re lated difference in body weights, macroscopic postmortem evaluations and organ weights in the lactating pups from Air Control animals. of this study

. After further review, only the kidneys of 1049M and 4044M were examined because they had gross findings, as per protocol. Table 1 on p. 25 corrected for the kidneys. animals.” To: “Microscopic examination of control and high

exposure group kidneys was not conducted in this study.”

In summary, a NOAEL of 2,000mg/m3 was determined based on the increased liver weights observed in the 10,000 and 20,000 mg/m3 exposed parental male animals. There was no effect at any exposure level on reproductive performance. Thus, with respect to reproduction performance, a NOAEL of 20,000 mg/m3 was concluded. I concur that these data support these conclusions. No co 

### m ment OK From report conclusions: 2K is the NOEL not
a NOAEL [which is OK to me]



### 211 (b) Research Group Reviewer

General Comments The report should have sequential numbering from start to finish. Will be done for final report. Ok ok All of the reports should have an appendix summarizing historical control data for reproduction endpoints. Hopefully this can be included for all of the reports. Appendix DD added. Ok verified ok

Specific Comments Page 5, 4th paragraph: Please replace "reasonably close" with "acceptably close," since there should be criteria for satisfactory comparison. Done although we do not have criteria for acceptance of this data. p.6 ok ok Please consider deleting the sentence beginning, "The exact cause of the differences ..." since this is

unnecessary. Done. p.6 ok ok Page 19, Temperature: The actual temperature range high end, 31.7C, seems quite hot. Please explain how long this excursion lasted and which groups were affected. Excursion lasted for approx. 6.5 hours and affected all groups. No change made to report at this time. Ok ok

Page 21, Section 2.12. I, Estrous Cycling: Please note the study days for the start of the daily vaginal smears. Done. Ok ok

Page 22, Section 2.12.4, Litter Evaluations, Observations: Please make note of the lung floatation test to distinguish stillborn and found dead pups, also that autolyzed remains found on the day of parturition were presumed to be stillborn. Done. Ok ok

### 211(b) Toxicology Research Group DIPE One-Generation Reproduction Toxicity Study Report Reviewer Checklist
PP CS LR checklist reviewers; RG response to comment;

7 7/27/2010 

### Comments Action/Response Page Lab response to comments

Page 31, Protocol Deviations, No. 6: Please provide the possible dispositions of the extra animals. Although the true disposition may not be known (or may simply not have been documented), it should still be clear that only humane options existed for their disposition. Added that “...The animals would havebeen euthanized or transferred to the inhouse colony.” Ok ok

Page 32, Section 3.1, Chamber Monitoring, 1st paragraph: The second sentence indicates that oxygen levels were at least 20%. I did not find a table with the chamber oxygen levels; please identify. Table VI in Appendix A indicates that the room oxygen level was 20°/0, but no values exceeded that. 

### Revised to “.....Chamber monitoring (see Table VI in Appendix A) showed that the chamber oxygen levels were
20%.....” p.33 ok ok

Chamber temperatures went as high as 28C; please indicate when this happened, if it happened to all groups, and how often. 

### The temperature only reached 28C on Exposure Day 62 for Groups 3-4 chambers. No change _ok ok

Last paragraph: Please consider revising "varied somewhat" to "varied within acceptable limits," since these variations were either within operating standards

or were not. Done. Ok bottom of p33 ok

Page 33, 2nd paragraph: Please consider revising "a reasonably close" to "an acceptably close" to indicate that operating standards

were met. Done. Ok ok

Page 33, Section 3.2, Mortality: This section ignores the humane sacrifices that occurred during gestation (No. 2536, Appendix E) and lactation (No. 4538, Appendix F). Since these are not mentioned in the gestation or lactation sections of the results, they should be mentioned here, and Appendices E and F should be referenced. Also, Table 1 does not indicate these humane sacrifices. These are noted in Tables 3 and 4; therefore these tables should also be referenced for the mortality section. Section revised for text and references. Looks OK Table also ok

Also, it should be noted that No. 2536, sacrificed on GD 1, is presumed to have been pregnant. This is too early for objective confirmation of implantation. Agreed but leaving as presumed pregnant, based on implantation sites, does not affect other parameters other than fertility index. ok

Page 36, Section 3.2.9, Parturition and Litter Survival: Please rephrase the last sentence; it reads awkwardly. Also, it would be helpful to have comments to explain why Group 1 lost so many more pups in the early postnatal period, as well as the early survival historical control range. Revised ok

Section 3.3.2, Pup Body Weight Data: Table 18 appears to include every possible interval for body weight gain. I don't think this makes the best This is limited by the TASC computer software and cannot be modified. Ok ok

### 211(b) Toxicology Research Group DIPE One-Generation Reproduction Toxicity Study Report Reviewer Checklist
PP CS LR checklist reviewers; RG response to comment;

8 7/27/2010 

### Comments Action/Response Page Lab response to comments

sense. Please consider reducing the number of intervals.

Page 37, Section 3.14.1, Terminal Evaluations: Please consider if the meaning regarding lungdiscoloration is that the findings are not treatmentrelated, or not adverse. There are greater numbers of discolored lungs in the exposed groups, which has also been observed in some of the other 211b reproduction studies. The lack of effects upon lung weight or histopathology ar e reasonable justifications for the position that the discoloration was not an adverse observation, but I'm not as convinced that this can be used to indicate that it wasn't treatment-related. Discussion revised to indicate treatmentrelated but non-adverse pattern of findings. Ok ok

Page 38, Section 3.4.3, Sperm Evaluations: Please correct the reference from Appendix BB to Appendix AA. Done. Ok ok

Page 39, Section 3.4.5, Pup Organ Weights: Table 22 has two page 1s; please handle as for other tables with a preface page. Also, in the evaluation of thymus weight, the variability of this parameter, as observed in the historical control range, might be useful for the brief discussion of thymus weight as unrelated to exposure. Table 22 will be properly paginated for the final report. HCD for pup organ weights added to the report in Appendix DD and the discussion in 3.4.5. Ok ok

Table 15 Please include a footnote to indicate that No. 2536, humanely sacrificed on GD 1, was presumed to be pregnant and included as such in the fertility totals. Tables 15 and 16 footnoted. Ok ok

Table 16, p. 2: The Group 4 number for mean pups delivered is shown as 13.1 here, but is summarized as 13.0 in Appendix R, page 4. Corrected. Ok ok

Appendix A There are two page 1s; please revise for continuous pagination. Also, each table begins with a Page 1. Appendix B is handled differently (continuous pagination throughout the appendix) in a preferable format. Will be properly paginated for the final report. Ok ok

Page 1 (second page 1), Temperature: Please indicate when 28C occurred and how long this temperature lasted. Page 6, section 2.3.2, Chamber sampling: Grammatical correction: "...t o characterized at least ..." (delete d). This was only seen on Exposure 62 for Groups 3-4.

Corrected. Same _no change

___________ Ok ok

Appendix O, Page 4: No. 4538, the humane sacrifice on LD 14, has a data value for feed consumption for the LD 1-28 interval. Data presented, but excluded from means/stats. Noted that excluded OK, but still weird that

### 211(b) Toxicology Research Group DIPE One-Generation Reproduction Toxicity Study Report Reviewer Checklist
PP CS LR checklist reviewers; RG response to comment;

9 7/27/2010 

### Comments Action/Response Page Lab response to comments

This needs to be removed. there’s a value for an animal that was dead during half of the interval.

ok Appendix P, page 4: No. 4540 appears to have a single 12-day cycle suggestive of pseudopregnancy. Please address. Footnoted and data excluded from means/stats. Ok ok



### 211(b) Research Group QA/QC Reviewer
A. Draft Final Report and Associated Raw Data Page 2, Compliance Statement: Since it was the sponsor’s responsibility to maintain the method of synthesis, fabrication, or derivation of the test fuel, and this has not been completed, it must be included in the sponsor ’s compliance statement. Corrected OK

Page 4, Quality Assurance Statement: Since HLS has inspected the sperm evaluation subcontractor (PAI) and is responsible for ensuring their GLP compliant status, these facility inspections should be listed on the QA statement for this study. Added by QA to the QA Statement OK

Page 6, Summary, last paragraph, line 4: Regarding the exposure-rela ted effects on male liver weight, a significant increase relative to brain weight was noted in the high-dose gr oup. This is discussed in the Results section (page 38) but is omitted from the summary. Should the summary be revised to include this information? Added to the Summary. OK

Page 9, Table of Contents: Table 18 includes pup body weight gain data, which is not indicated in the table title presented in the Table of Contents. Should the Table of Contents be revised to indicate that table summarizes both mean pup body weight and weight gain data? Corrected OK

Page 13, Section 2.3, Experimental Outline: Footnote “b” for this table indicates histologic examinations were perform ed on reproductive tissues for the randomly selected control and high-exposure animals. Additional protocol-required tissues (adrenals, lungs, pituitary and lesions) were also examined. It is recommended that the table be revised to include this information. Table footnote revised to include that ‘select’ tissues were also examined. OK

Page 16, Section 2.5.7, Weight at Initiation of Exposure: The protocol on page 11 allows for StudyDirector discretion in accepting the outOk

### 211(b) Toxicology Research Group DIPE One-Generation Reproduction Toxicity Study Report Reviewer Checklist
PP CS LR checklist reviewers; RG response to comment;

10 7/27/2010 

### Comments Action/Response Page Lab response to comments

The weight ranges for the parental males (186 to 245 grams) and females (130 to 179 grams) were not strictly within the protocol specified ranges of 150 to 200 grams for males and 125 to 175 grams for females. These departures from protocol specifications should be addressed in the data and report. of-range body weights and we do not therefore consider this to be a protocol deviation.

Page 18, Water Analysis: The report text states that analyses of facility water samples for chemical and microbiological contaminants are conducte d biannually (i.e., twice yearly). However, the water analysis reports from Lancaster Laboratories that are included in Appendix BB of the draft report present results from samples collected on September 25, 2000 and March 27, 2002. These sampling dates do not reflect biannual analysis, nor do they closely bracket the dates of in-life study conduct (May – September, 2002). Please correct/clarify water analysis information as appropriate. The assays have been replaced with more timely reports from March and August
2002. ok

Page 23, Section 2.13.1, Parental Animals, Necropsy Information: Regarding the termination of P0 males, the report text states that the males were euthanized after the last day that the F1 litters were delivered, whereas the protocol (Section 13.1.1.3, page 21) states that males were to be euthanized proximate to the date that the last litters were weaned. The data indicate that males were terminated on September 10 and 11, 2002, which was actually one to two weeks prior to the weaning of the litters. The report text needs to be revised to accurately reflect the termination schedule for the parental males. Additionally, the termination of the males prior to the weaning of the last litters should be addressed as a departure from the protocol in the data and final report. Protocol corrected in Amendment #1 which is included with the 2nd Draft report to be signed by the Sponsor.

Report text is correct as is. P0 males were euthanized after the last litters were delivered. Ok – amendment1 is

not in draft report

AmendI received as separate document; requires signatures

Page 24, Section 2.13.1, Parental Animals, Tissues Preserved and Examined Histopathologically: The third sentence incorrectly states that male reproductive tissues were examined for all P0 control and high exposure animals. The pathology data reflect that these tissues were examined from only the 10 randomly selected control and high dose males. The text should be corrected to reflect actual study procedures. Corrected. Ok

Page 25, Section 2.13.1, Parental Animals, Tissues Preserved and Examined Histopathologically, Table 1: Table corrected. ok

### 211(b) Toxicology Research Group DIPE One-Generation Reproduction Toxicity Study Report Reviewer Checklist
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11 7/27/2010 

### Comments Action/Response Page Lab response to comments

The table incorrectly indicates that brain, kidneys, liver and spleen were examined microscopically from the Group I and IV parental animals. These tissues were preserved but were not microscopically examined. Table 1 should be revised to reflect actual study procedures. Page 26, Section 2.13.2, F1 Pups, Method of Euthanasia: The text states that after Lactation Day 4, pups were euthanized by exsanguination following carbon dioxide anesthesia. This statement is not true for the pups from Litter No. 4538, which were sacrificed on Lactation Day 14, following the humane sacrifice of their dam. The data indicate that these pups were euthanized by IP injection of sodium pentobarbital. The text should be revised to reflect this exception. Also, the method of sacrifice for this litter should be addressed as a departure from protocol (see sections 13.1.2.1 and 13.1.2.3) in the data and final report. Section revised to state that F1 pups culled or sacrificed prior to weaning were euthanized by IP injection of sodium pentobarbital; weanlings were euthanized by exsanguination following CO2 anesthesia.

Protocol deviation added to data and report. Ok

Page 26, Section 2.13.2, F1 Pups, Dead and Culled Pups: The report text states that partially cannibalized pups were discarded after examination. According to the study data, two partially cannibalized pups (2538-04 and 3534-02) were apparently saved. The report text should be revised to reflect this exception. Also, this should be addressed as a departure from protocol Section 13.1.2.3, which states that partially cannibalized pups were to be discarded. Text corrected to indicate cannibalized pups were discarded or preserved.

Protocol deviation for 2538-4 added to data and report.

Per review of saved tissues, Pup 3534-2 was not saved. No deviation necessary. Ok

Page 28, Section 2.15.1, Statistical Analysis of Continuous Data: The report text and summary tables indicate that estrous cycle data were analyzed as continuous data. However, the protocol (Section 15.1, pages 26 and 27) does not include estrous cycle data in the list of continuous data to be analyzed. It appears that the protocol should have been amended to include the analysis of these data. This will be included in Amendment#1 to be provided in the 2 nd draft report. Ok – amendment 1 is not in this draft

Page 30, Section 2.15.3, Incidence Data Analysis: Should this paragraph be revised to indicate that incidence data were first analyzed by Chi-square test followed by Fisher Exact test if the Chi square test was significant (as per protocol section 15.2.1)? This will be included in Amendment#1 to be provided in the 2nd draft report. Ok, amendment 1 is not in this draft

Page 31, Protocol Deviations: Excursions from the protocol-specified animal room temperature and humidity ranges were noted throughout the course of the study. It is recommended We do not report animal room temperature and humidity ranges excursions as protocol deviations based on the protocol wording of ‘maintained Ok - all

### 211(b) Toxicology Research Group DIPE One-Generation Reproduction Toxicity Study Report Reviewer Checklist
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### Comments Action/Response Page Lab response to comments

that this be included in the deviations section of the final report. See also items 6, 8, 11, 12, 13, 32 and 38 of this audit report for additional protocol departures that may need to be incorporated into the Protocol Deviation section of the final report. to the maximum extent possible’.

6 – see previous response (Page 16, Section 2.5.7, Weight at Initiation of Exposure)

8 - see previous response (Page 23, Section 2.13.1, Parental Animals, Necropsy Information)

11 - see previous response (Page 26, Section 2.13.2, F1 Pups, Method of Euthanasia)

12 - see previous response (Page 26, Section 2.13.2, F1 Pups, Dead and Culled Pups)

13 - see previous response (Page 28, Section 2.15.1, Statistical Analysis of Continuous Data)

32 – No protocol deviations noted. (Appendix B, Analytical Report, Page 22-23, 2,3-Dimthylbutane, all samples)

38 – No protocol deviations noted. (Appendix O, Individual Maternal Lactation Feed Consumption) Page 36, Parturition and Litter Survival Data: The results section does not address pup sex ratios. Should this section of the text be revised to mention that this parameter was also unaffected by treatment? Section revised to also discuss sex ratio. Ok

Page 38, Section 3.4.3, Sperm Evaluations: The reference for the sperm evaluation report should be Appendix AA (not Appendix BB). Corrected. Ok

Table 1, Summary of Survival and Pregnancy: The summary table indicates that one pregnant female in Group I and Group III died or was sacrificed postpartum (incidences of 1 are reported under “Pregnant, Died/sacrificed postpartum” and under “Total females died/sacrificed” for each of these groups). Appendix C (Individual Animal Termination History) and the original data reflect that all females in these two groups were terminally sacrificed. These apparent discrepancies should be corrected/clarified in the report. Table 1 corrected. Ok

Table 1, Summary of Survival and Pregnancy: ok

### 211(b) Toxicology Research Group DIPE One-Generation Reproduction Toxicity Study Report Reviewer Checklist
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The summary table indicates that no pregnant females in Group IV died or were sacrificed postpartum(incidences of 0 are reported under “Pregnant, Died/sacrificed postpartum" and under “Total females died/sacrificed”). Appendix C (Individual Animal Termination History) and the original data reflect that Dam No. 4538 was a humane sacrifice on Lactation Day 14. Should this be included in the summary data presented in Table 1? Please correct/clarify as appropriate. Corrected.

Table 3, Within Normal Limits entry: These counts of normality include the females that were not pregnant. Should they be summarized with the animals that ended up delivering and completing the study? Please comment. We agree it is OK and the TASC program tabulates in that manner. Ok

Table 4, Terminal Sacrifice entry: for the Controls, Group 3 and 4 entries may not be accurate. Group One Animal 1549 was not entered as a terminal sacrifice in Appendix F, so was not summarized here. The same is true for Animal Number 3534. For Group 4, I count 22 animals at terminal sacrifice from Appendix F. Please clarify. Both animals were sacrificed on Day 30, therefore were not presented on this table (range was LD 1 to 28). Table re-printed with range set to LD 1 to 30. Ok

Table 16, Summary of Delivery and Litter Data: Pup death and survival data on pages 1 and 2 and pup sex ratio on page 3 of the summary table are reported through day 21 of lactation. Shouldn’t these data be reported for the full 28-day lactation period? No changes possible due to TASC system limitations. Ok

Table 16, Summary of Deliver y and Litter Data versus Appendix R, Individual Delivery and Litter Data, Sex Ratio on Day 0: Appendix R indicates that there were a total of 155 male pups on Day 0, whereas Table 16 reports a total of 150 male pups at this interval. Is this due to the exclusion of 5 male pups from Litter No. 4538, which were terminated early due to the humane sacrifice of the dam? Please address/clarify as appropriate. No change necessary. Appendix R clearly states that Litter 4538 is excluded. Ok

Table 17, Summary of Pup Clinical Observations During Lactation: The table title indicates that incidences are presented as “frequency/animals”. It appears that data are actually presented as “frequency/number of affected litters”. No change to table title possible due to TASC system limitations. Ok

Table 20, Summary of Pup Necropsy Observations versus Appendix W, Individual Pup Necropsy Observations: Appendix W reports extern al findings of “domed head” for Pup Nos. 3531-5 and 3531-13. These observations do not appear to be summarized in Table No changes necessary. Table 20 excludes External Exam findings as these are confirmation of In-life findings. ok

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### Comments Action/Response Page Lab response to comments

20. Please correct/clarify as appropriate. Appendix A, Inhalation Report, Table V, Miran Calibration: The Average Absorbance (V olts) for the last two concentrations should be 0.639 and 0.777. Are these typographical errors or were incorrect values used for the regression analysis? Please clarify. The data is correct and the report has been corrected. Ok as long as correct value is reported

Appendix A, Inhalation Report, Pages 1 and 2: Some mention as to the duration or frequency of the temperature and humidity excursions should be given. Frequency of excursions added. Ok

Appendix A, Inhalation Report, CMR Table, Page 11: For the chamber concentrations on 8/25/02, it is not clear where the value of 3110 mg/m3came from. The first concentration noted was 1410 and then 5 minutes later the value of 3520 was recorded. Five minutes after that the value was 2700.Is 3110 a time-weighted average? Please clarify. Per SOP, the D1 and the D2 samples were averaged and the original value was ignored as an unconfirmed outlier. Ok

Appendix B, Analytical Report, Page 5, Results and Discussion: A statement summarizing the results is needed. It should be indicated that no drift in the major component proportions was noted throughout the study. Added. Ok

Appendix B, Analytical Report, Page 5 Results and Discussion: There should also be some discussion about the amount of DIPE that was seen in the vapor. For some of the exposures (example - Exposure 84), a different column was used that se parated the N-hexane and DIPE showing around 3 Area Percent N-hexane and 18 or 19 Area Percent DIPE. This is consistent with the nhexane content of the other test materials in thisprogram showing between 2and 4 Area Percent Nhexane. Added to the results and discussion section. Ok

Appendix B, Analytical Report, Page 15, 2,3- Dimethylbutane, Sample 3008: Please review the values for this component. It appears that the peak may have split and the retention time shifted. The value appears correct on the GC print-out. Correct as is. The sample peak was reidentified on the GC print-out. Ok

Appendix B, Analytical Report, Page 22-23, 2,3- Dimthylbutane, all samples: A different column was used and it appears that the peak may have split here as well, but wasn’t added to the main peak. Please verify. Peak areas of split peaks were added on the data and the report was updated. Ok

Appendix D, Animal number 1540: This animal was a terminal sacrifice on Day 108, not Animal’s death is correctly entered as Day 108, however, because this was not a ok

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105. How could it have been sacrificed on the computer on Day105? The same is true for animal 3530. Please clarify. As well, on Table 2, it is not entered as a Terminal Sacrifice for Day 105. It doesn’t show up until Day 112. Please also verify that animal 2543 was sacrificed when appropriate and that it was summarized correctly. scheduled day for clinical observations, it does not appear on the report on the actual day of death. Summary report (Table 2) presents deaths occurring between scheduled periods at the next interval; individual data report (Appendix D) presents this death on the previous interval. Notations have been added to the preface pages. Appendix E, Animal number 2536: An observation of “decreased FV” is entered for this animal without a corresponding footnote explanation. Abbreviation removed and replaced with full text observation (fecal volume). Ok

Appendix F, Page 7, Animal No. 3534: This animal was not observed at the required intervals and appears to have not been observed at termination(appendix F). Please include this as a protocol deviation and verify that the data has been reported correctly, in summaries if appropriate, or separately if not. No deviation necessary. Animal lost its litter and was placed on a weekly observation schedule. See also Animal
No. 1549.

Both animals were sacrificed on Day 30. Appendix extended to Day 30 to show Terminal Sacrifice. Ok

Appendix N, Individual Gestation Feed Consumption: For Group I female1531, please recheck the reported food consumption value for Gestation Days0-4. Appendix N reports consumption of 78 grams, whereas the raw data appears to reflect food consumption of 85.6 grams (572.2 grams offered on Day 0 and 486.6 grams remaining on Day 4). Please recheck and correct/clarify as appropriate. No change to report. Food consumption is reported as gram s of food consumed per kilogram body weight per day: 85.6 g consumed/0.274 kg body weight/4 days = 78.1 g/kg/day. 0k

Appendix O, Individual Maternal Lactation Feed Consumption: For Lactation Days 4-7, Group I females 1535 and 1542 are reported in the appendix shaving invalid weights due to the amount of feed remaining being greater than amount fed. The study data indicate that these weights were excluded because the animals were not able to access their water valves due to faulty caging. The report appendix should be corrected to reflect the actual reason for the exclusion of these food consumption values. Appendix corrected. Ok

Appendix O, Individual Maternal Lactation Feed Consumption: For Lactation Days 14-21, Group IV female 4530 is reported in the appendix as having an invalid weight due to the amount of feed remaining being greater than amounted. The study data indicate that the weight was excluded because of technician weighing error (technician forgot to tare jar with old feed prior Appendix corrected. ok

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to adding new feed). The report appendix should be corrected to reflect the actu al reason for the exclusion of these food consumption values. Appendix R, Individual Delivery and Litter Data: For Female No. 4530, the abbreviation “pc” is used in the “Duration of Gestation” column. This abbreviation is not defined in the notations at the bottom of the table. Abbreviation was not necessary and was removed. ok

Appendix S, Individual Pup Sex and Status During Lactation and Appendix W, Individual Pup Necropsy Observations: Pup No. 2538-4 is reported as being found dead on Lactation Day 1. The study data (Individual Dead Pup and Internal Examination Record) indicates that this pup died on Lactation Day 0 (August 23, 2002). An edit dated September 25, 2002 shows that the status and death day were changed from missing on day 1 (4/1) to dead on day1 (1/1), with the reason for the edit being “per dead pup sheet”. It is not clear, however, why the day of death was changed from Lactation Day 0 to Lactation Day 1. Please correct/clarify as appropriate. This correction may affect associated summary tables. Data reviewed. It appears the day ofdeath edit was made in error. Data reeditted to day of death = Day 0. Associated tables and appendices reviewed and revised as necessary. ok

Appendix W, Individual Pup Necropsy Observations: Appendix and study data reflect that lung flotation test was performed for Pup No. 3534-1 at the time of its death. Since this pup was found dead on Lactation Day 1, rather than Lactation Day 0, it is not clear why lung flotation was performed . The protocol required lung flotation only for pups found dead at birth. Please clarify in study data and report why lung flotation was performed. Protocol deviation added to data and report. ok

Appendix Y, Individual Pup Organ Weights and Organ/Body Weight Ratios: The weights reported in the “final body weight” column of this appendix are the Lactation Day 21 body weights rather than the Lactation Day 28 final weights. All values need to be edited to reflect the Day 28 values and the organ/body weight ratios recalculated using the Day 28 values. The associated summary tables with statistics appear to report ratios calculated using the correct (Day 28) weights, but this should be rechecked to verify. Corrected. ok

Appendix Z, Individual Animal Gross and Microscopic Observations: For Group I female 1544, the reported gross observation for adrenals indicates red discoloration. The raw data printout dated September 20, 2002 does Edit dated 20 Sep 02 added “Red” to this finding. No change necessary (see also Appendix V). ok

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not specify a color. Was this observation edited to include the color modifier "red”? Please address in data and/or report as appropriate.
B. Additional Raw Data and Protocol Comments Study Protocol, Section 13.1.2.3, Dead and Culled Pups: The protocol states that culled F1 pups will be examined externally and if unremarkable, will be euthanized and discarded. The study records (Individual Pup Necropsy Observations) appear to indicate that culled pups were examined, at necropsy as an entry of “No Remarkable Observations” is documented for each culled pup. Please document as protocol deviation or otherw ise clarify as appropriate. Not a protocol deviation since culled pups were examined as per protocol requirement. ok

Analytical Data: No standard runs were incl uded with this study data. They are probably included with the DIPE Subchronic Study. This needs to be clearly referenced. As well, no certificates of analyses were present for standards. Correct as is: test substance standards are present for all runs in this study. See injection #003 and #009 in each run. ok

Analytical Data: There are system calibration data for study 6129 (10/10/01),6130 (2/4/02) and 6131 (6/6/02) and then print-outs for 6131 dated 8/22/02 included with study 4205 data. It is not clear what these calibration runs are for and why they are included with study 4205 data. Are they analyzing a butane standard, and shouldn’t they be included with their respective data and referenced in this data book? Please clarify. System calibration data from other studies removed from 00-4205. ok

The data form entitled “Tissues Saved Required by Protocol ”for Group I males 1039 through 1051 does not include spleen in the list of tissues saved. Please correct the form to indicate that this protocol-required tissue was retained for these animals. Clarified in data that spleens were saved for 8 of these males but not for 5 of these animals. This has been noted in data and report as a protocol deviation. ok

The Individual Dead Pup Internal and External Examination Record for Pup No. 3550-1 indicates that that this dead pup was examined on Lactation Day 1(August 22, 2002) and determ ined to be stillborn. Since the dam initiated delivery on August 21 but did not complete until August 22, 2002, this pup is reported as a Day 0 stillborn, although the data still reflects it was examined on Day 1. It is recommended that a notation be added to the “Dead Pup Examination Record” to clarify the pup’s status. Data has been annotated to clarify the pup status. ok

In Book 1 of 1, the edit record for the terminal estrous cycle data (hand recorded data) dated November 1, 2002 does not indicate the interval being edited. Please add this information to the edit record. These edits refer to the addition of estrus parameters to the necropsy portion of the Xybion protocol and study list so that the terminal smear readi ngs could be entered. ok

For Gestation Body Weights, the handwritten Data was corrected 24Jun04. ok

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correction sheet for Group IV female 4549 should be corrected to indicate “Gestation Day 4” rather than “Lactation Day 4” for the data being corrected. For Group III male 3047, an initial day 112 body weight of 522.1 grams was recorded. Upon recheck, the weight was determined to be 542.9 grams and this weight was reported. According to SOP TX-02-5.2, a second verification weight should have been taken since the first verification weight differed from the initial weight by more than 20 grams. This should be addressed as an SOP deviation in the study records. SOP deviation memo was signed 23June04 and added to data. ok

It is not clear from the data whether the carcass of Pup No. 1528-7 that died on Lactation Day 2 (August 23, 2002) was saved. This pup was noted as being autolyzed at the time of examination and is not listed on the “Dead Pups Saved” form. Please clarify in data whether this carcass was saved or discarded. If the carcass was not retained, this will need to be documented as a protocol deviation (see Protocol Section 13.1.2.3 which states that dead be retained). Clarified in data that tissues were saved. ok