### 211(b) Toxicology Research Group Gasoline DIPE VC: A 13- Week Whole Body Toxicity Study in Rats with Neurotoxicity

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### Comments Action/Response Page

EPA

Overall, the NHEERL reviewers find that the methods employed were in accordan ce with the guidance of the 211(b) test rule.

We provide the following comments: Noted.

Genotoxicity. The genotoxicity assessments included a Rat Peripheral Lymphocyte Sister Chromatid Exchange Assay

and a Rat Micronucleus Test. These were conducted in

accordance with the testing guidelines and no technical

weaknesses were identified. The results from these studies were negative. Noted. Vol IV Appendix X micronucleus; Appendix Y

SCE

Immunotoxicity . The assessment of immunotoxicity was also carried out appropriately and in accordance with testing guidance. The results of the immunotoxicity evaluation showed a suppression of the antibody response of the humoral immune component of the immune system of animals treated with 20,000 mg/ m 3 . The results of this assessment do not appear in the executive summary of the report or the main text describing the overall study results in

Volume 1. Instead the reader is referred to the immunotoxicity report of the results of this study. This is toxicologically significant finding and should be included in the executive summary of the main report. The results of this and all satellites studies have been added to the report summary in

tabular form.

Accepted. The summary and main report only cover the routine sub-chronic toxicology parameters as well as detailed evaluations of neurotoxicity parameters (with the exception of the glial fibrillary acidic protein (GFAP) evaluations). The GFAP, genotoxicity and immunotoxicity reports, which include endpoint specific NOAELs, are appended to the main report. Vol 1 p.8

Vol IV Appendix Z Immunotoxicity

Neurotoxicity. The separate report on glial fibrillary acidic protein (GFAP) fulfills the requirements of the test rule. No adverse changes were observed in GFAP levels as a function of treatment. The main report (Volume 1) contains data from the functional observational battery (FOB) and the assessment of motor activity.

 Motor Activity. There is an error in the reporting of motor activity results. On page 51 of the report it is stated that there was a statistically significant difference in the motor activity levels of female rats in “study week 4”, but according to the statistical report and data tables this effect was observed in study week 8. These motor activity changes were not dose-related or consistent over time, and

therefore are not considered to be adverse effects of treatment with the test compound.

 FOB data: There is a statement in the summary and the statistical report sa ying that there were only

6 of the measures on the discrete or nominal scales with “any variation in response”. This is incorrect. For example, in addition to the 6 variables mentioned, there is also variability in data for visual approach, palpebral closure, lacrimation, pinna reflex, chromodacyorrhea, gait, locomotion

and arousal. This statem ent should be corrected. R&D corrected to week 8

Accepted

OK as is - Statistical report and R&D states that no other variables had ‘meaningful’

variations.

Accepted. After reviewing EPA’s comments under, FOB data , the RG agrees with the study director’s interpretation of the results. Sect 3.7.1 p.52

Sect. 3.7.2 p.52

### 211(b) Toxicology Research Group Gasoline DIPE VC: A 13- Week Whole Body Toxicity Study in Rats with Neurotoxicity

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### Comments Action/Response Page

Peer Reviewer – Schlesinger

General Comments The study was conducted in the proper manner and the results presented support the conclusions reached. There were no protocol deviations that would have adversely affected the study outcome. Noted.

There needs to be consistent criteria for data evaluation, especially in terms of level of significance for all aspects of the study. For exampl e, the micronucleus study used 0.01 as the significance level, while other studies uses 0.05, and the main study used both. Each lab used different criteria for statistical significance based on their usual practices. In the future, this criteria can be set in the Testing Facility protocol to be consistent for

all involved labs. Accepted Main Report p.6_Summary _ The sentence concerning reasons for

differences between measured and nominal concentrations

should be deleted from this section as it is not mentioned in the main body of the text and, in fact, the correspondence is quite good. Deleted as suggested.

Accepted Vol.1 Summary p.6

2.11.5 p.23 Administration of Test Substance_ The group numbers are inconsistently indicat ed as I, II, III, IV in the text and then 1, 2, 3, 4 in the Tables. This is limited by the software in use at the time of this study and cannot be edited. Accepted Sect 2.11.5 p.24

2.20 p.42 Statistical Analysis _ It is noted that each time interval was analyzed independently. The investigators should indicate why they did not use a 2 way ANOVA with time as one of the parameters. This is limited by the software in use at the time of this study.

Accepted Sect. 2.20 p.43

It is noted that a number of different post hoc test may have been used for both parametric and non parametric analyses. What determined which test was used for a specific data set. This was established by the specifics of the statistical software package in use at the time of this study. Accepted Why were both 1% and 5% used as levels of significance rather than a single level selected prior to the study. This was established by the specifics of the statistical software package in use at the time of this study and as per the protocol. Accepted Sect 2.20 p.44

What is meant by the statement that separate analysis may have been done by sex (replace with gender) to “explain the

nature of the effect.” The statistician may have separated the analysis by sex in order to look at the trends for each sex to interpret the data best. Accepted. Sex was not replaced by “gender’ Sect 2.20 p.44

MA and FOB

3.1 p.48 Chamber Monitoring _ It is noted that some particles are invariably occurring in chamber air. However, if air incoming to the chambers is properly filtered, there should be none. The investigators should address this issue. The incoming air was unfiltered room air and as such a low concentration of particles is always present. Explanation adequate. Report does not

indicate room air was “unfiltered” either in Methods [Administration sect 2.11.5] or3.1 Sect 2.11.5 p.24 Sect 3.1 p.49

3.4 p.50 Body Weights_ The females in Group III seem to show a clear trend towards greater body weights beginning at about week 3. While this may not be in a dose response pattern, the consistency in this group could suggest that something is different about these animals. This just seems to be an anomaly; also the standard deviation for this group is similar to the other groups since there are no remarkable outliers in the group. Accepted Sect 3.5 p.51

### 211(b) Toxicology Research Group Gasoline DIPE VC: A 13- Week Whole Body Toxicity Study in Rats with Neurotoxicity

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### Comments Action/Response Page
3.6 p.50 Feed Consumption _ Both males and females seem to show an increase in consumption beginning in week 14 and continuing until the end of exposure. This should be

noted. [Note: This comment refers to untreated recovery period weeks 14 through 17 – no exposure] Agreed and added additional discussion regarding the increase in feed consumption during the recovery period.

Accepted Sect 3.6 p.51

3.7.1 p.50 Motor Activity_ Is the response difference between low and medium exposure groups noted here in the pretest motor activity data or in the data obtained during exposure? This is not clear. Al so unclear is the basis for the statement that there is an increase in activity in the females at mid-exposure during the 4 th week. As explained on page 3 of the statistical report by Mark Nicolich, the response difference is a collation of all intervals during the study. The actual increase was in week 8

and the R&D has been corrected. Accepted Sect 3.7.1 p.51-

52.

Statistical report Table 7

3.8.2 p.52 Coagulation Time_ Table 11 shows what appears to be an increase in mean coagulation values in all animals exposed to the DIPE vapor. While there may not be a dose-response relationship, the occurrence of the effect in all exposed groups should be addressed. Additional discussion added to this section.

Accepted Sect 3.8.2 p.53

3.8.3 p.52 Clinical Chemistry_ A number of indices did show consistent changes for all or some of the exposure groups, e.g., CK in males, SDH in males, at week 4. If there are changes at week 4 but not 13 should this be interpreted as being not significant? Additional discussion added to this section but overall these findings assessed to not being of toxicological significance.

Accepted Sect 3.8.3 p.53

3.9 p.53 Organ Weights_ There appears to be a decrease in lung weight at the highest exposure level in males. This

should be addressed. Added to the discussion in this section.

Accepted Sect 3.9 p.54

Immunotox Report This is a well-written report. The conclusions are supported by the data presented. Noted. Vol IV Appendix Z Data p13 This is an excellent, clearly written description of the statistical assays used and the criteria for accepting changes as statisti cally significant. Noted.

Organ Wts p13-15_ While it is true that the only statistically significant change in spleen weight occurred at the low dose, all of the exposure groups showed greater relative weight than did the air control. This was not seen for the thymus, so perhaps there is a real effect on the spleen.

The investigators’ note that the “effect of DIPE on spleen weights and spleen cell number s cannot be appropriately evaluated since the animals in this study were sensitized with sheep erythrocytes.” If it is not possible to provide any conclusion as to the effect of the test agent on these parameters, then why were these assays performed at all in this study. Table 2 is missing a table number at the top of the page. A more appropriate evaluation would have been obtained using unsensitized animals. However, unsensitized animals were not available in this study. Futhermore, as demonstrated by the decrease in both spleen weight and spleen cell number of the CPS group, even in immunized animals, potent immunosuppressive compounds do have the ability to suppress these endpoints. Since the purpose of this study was to determine the potential immunotoxicity of DIPE, it was germaine to collect this data and evaluate it under the caveat discussed previously. Had DIPE been a potent immunosuppressive agent we would have anticipated seeing decreases in spleen weight and spleen cell numbers. We recommend deleting the 2 sentences since we no longer include these statements in reports for studies that only have immunized

animals. Accepted

### 211(b) Toxicology Research Group Gasoline DIPE VC: A 13- Week Whole Body Toxicity Study in Rats with Neurotoxicity

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### Comments Action/Response Page

The number has been added to table 2. Accepted

SCE Assay Report The conclusion is supported by the data presented Noted. Vol IV Appendix Y p.11 Evaluation of Test Results_ This reviewer feels that the criteria used by the PI as guidelines in evaluation are flawed. In my opinion, the finding of a statistically significant change at only the highest exposure level would not be suspect but, rather, could likely indicate that some exposure threshold for response had been reached. There do

not need to be effects at lower le vels for an effect at a higher level to be biologically ”real”. This criteria has been used and accepted for all other reports in this 211(b) test program and will not be modified for this study. Accepted Reviewer’s disagreement on criteria for a positive result is of no consequence in this stud y as no increases in SCE frequency were seen at any dose level and no statistical significance observed p.12

GFAP Study This is a very well written report. The conclusion is supported by the data. Noted. Vol IV Appendix AA p.17 Statistics_The name of the post hoc test used should be provided. Added to the report. Accepted – Least significance difference test [Keppel, 1973] p.17

Micronucleus Study The conclusion is supported by the data presented. Noted. Vol IV Appendix X



### Peer Reviewer – Goldsworthy
The potential in inhalation toxicity of G-DIPE was assessed via whole body exposures to male and female rats for 13 weeks followed by a 4-week recovery period. In this report, standard toxicology and neurotoxicity parameters were presented (genotoxicity and immunotoxicity results were presented in separate reports ; reviews presented below). The design for the study was appropriate and sound and the study was properly conducted. The report was clear and comprehensive. The deviations noted had no impact on the study conclusions or data integrity. Exposure techniques and procedur es were adequate to test the potential toxicity of gasoline-DIPE. Exposure levels of test substance were relatively close to target levels. Animals were exposed to all major components of gasoline-DIPE in their expected proper propor tions. Week to week data consistently was noted, indicating stability of test substance and proper techniques. Noted

Experimental data revealed no effect of G-DIPE on survival, feed consumptions or gross abnormality at necropsy. Red nasal discharge was observ ed in the 20,000 mg/M 3 exposed animals; these observations ab ated following the 4-week recovery period. p. 49 Text should note when the red discharge was first

observed. Timing of the observation added to the R&D and Summary

Accepted Summary p.6 Sect 3.3.2 .p.50

No exposure –related effect was observed on functional

### 211(b) Toxicology Research Group Gasoline DIPE VC: A 13- Week Whole Body Toxicity Study in Rats with Neurotoxicity

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### Comments Action/Response Page
observation battery; no effect was observed with opthalmoscopic assessments. Some differences in motor activity were noted in the low and medium exposure groups; due to a lack of consistent change among dose levels, no exposure-related changes were considered to be associated

with G-DIPE. p.6 , 2 nd paragraph, last sentence Summary should note the statistical significant findings observed and the rationale for concluding that these effects were not exposure related. Not necessary to detail in Summary but

discussed in the R&D.Sect 3.3.2 Accepted Motor activity p.44

There were no observed exposure-related effects in coagulation values at 4-week an d terminal intervals. High dosed animals exhibited decreased reticulocyte counts at 4- week, terminal sacrifice and recovery intervals.

At 4 weeks, statistical decreases in reticulocyte counts were also observed in male rats –text on p.51 (bottom) and in Summary (p.6, bottom) should replace “Very slight (13- 17%) decreases” with “statisti cal (13-17%) decreases”. Additionally, conclusion statements (p7 and p.55) should clarify that decreases in reticulocytes occurred at all dose

levels at 4 weeks. Noted

Revised Summary and R&D for “statistically significant (13-17%) decreases” and added

intervals to the conclusion statements.

Accepted – the decrease in reticulocyte counts in males at all doses in noted on p.6 and p. 53. The NOAEL is based on the consistent decrease in reticulocytes at 4 weeks, 13 weeks, and recovery in the 2000 mg/m3 group. Summary p.6 Sect 3.8.1 p.53

Sect 4.0

Conclusions p.56

It is debatable whether this adverse effect should be considered in the NOAEL setting; interestingly, other API sponsored studies (i.e. Gasoline ETBE) also demonstrated a treatment effect on reticulocyte counts. In the Hematology section (p 52) and Summary (p.6-7 ), text states that “similar

differences” were not seen in female rats. While this is true, the report failed to note potential decrease observed at 4-week and recovery time points in absolute monocyte values. Discussion of monocyte decreases added to Summary and R&D.

See above See above

P52 and p7 Clinical Chemistry is incomplete and does not adequately present the data. On p.52 ad well as p7 (Summary) state “no exposure-related

differences in clinical chemistr y values in the test substance male animals...” It is not clear why test only refers to male rat assessments. Furthermore, data (Table 12) show some consistency in the enzymes affected and thus it appears that there is a likelihood that a few changes were treatment related. For example, SDH values are statistically decreased in the mid and/or high exposure groups in male rats a 4-week and recovery time points, and in female rats at 4-week, terminal and recovery groups. Similarly, LD values are statistically decreased in male rats at 4-week (all exposure levels) and recovery and in female rats at terminal and recovery. Discussion of clinical chemistry findings revised as suggested in Summary and R&D.

Accepted Summary p 7 Sect 3.8.3 p.53

### 211(b) Toxicology Research Group Gasoline DIPE VC: A 13- Week Whole Body Toxicity Study in Rats with Neurotoxicity

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### Comments Action/Response Page
Text (both Results and Summary, p.7) should be edited and be more comprehensive in noting these changes. It is appropriate to not consider th ese defects adverse since no histopathology changes were noted and decreases were relatively small. No exposure-related differ ences were noted in gross abnormalities, organ weights and brain measurements. Microscopic findings were only present in the kidneys of male rats. These renal findings were consistent with hydrocarbon-induced nephropathy; this species and gender specific alteration is generally not considered relevant for

human risk assessment. Noted. Sect 3.10 Pathology Kidney p.55

For clarity, provide report titles for the associated study reports referred to on p.34,35 and 36. Currently, they are referred to as “BioReliance Report”,” Huntingdon UK Report” and “Immunotox Report.” OK as is per the accepted text for all prior reports in this test program. Accepted – These studies are identified in the

Table of Contents in Vol IV Immunotox Report This study evaluated the potential of 4 weeks of exposure to Gasoline-DIPE to affect the humoral immune component of the immune system when evaluated by antibody-forming cell response to T-dependent antigen sheep erythrocytes. This study was conducted properly and was scientifically

sound. Data demonstrate that Gasoline-DIPE did not affect terminal body weights, spleen or thymus weights; the increase in relative spleen weight at 2000 mg/M 3 was appropriately noted as not biologically significant. Gasoline-DIPE Vapor Condensate did result in a dose related decrease in the spleen IgM andtibody-forming cell response to sheep erythrocytes. This affect was only statistically significant at the high exposure level, although the mid exposure group (10,000 mg/M 3 ) exhibited decreased values as compared to controls. Text (p.16) notes that vehicle animals were “somewhat lower than that had been observed in the past...”; it would be useful to present historical values. Without this information it is difficult to ascertain if the mid exposure data would be a significant finding on a study with ‘normal vehicle values’. The positive control CPS resulted in the anticipated results, verifying proper assay conditions. Since the study was conducted using outbred animals, the historical control values provide limited help in interpreting the response. Each study needs to be interpreted within the context of the animals used in the study. For example, our historical control data for AFC/10^6 study means ranges from 286-

4167 for female SD rats. Accepted- Response supplied by Dr. Kimber White, study director

The sentence has been modified to “While the overall response of the study was somewhat

lower....” Accepted Vol IV Appendix Z

p. 16

The conclusion that, under the experimental conditions of this study, Gasoline-DIPE did adversely affect the functional ability of the humoral immune component of the immune system accurately reflects the st udy results. This conclusion, as stated on p.9 and p.17, could note at what exposure level the decrease occurred, and st ate a NOAEL of 10,000 mg/M 3 This has been added.

Accepted p.9 and p.17

SCE Assay Report The study is properly conducted and scientifically sound. The positive controls fulfilled the requirements for a valid test. The report correctly concludes that Gasoline-DIPE Vapor Condensate was negative for the induction of SCEs in Noted. Vol. IV Appendix Y

### 211(b) Toxicology Research Group Gasoline DIPE VC: A 13- Week Whole Body Toxicity Study in Rats with Neurotoxicity

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### Comments Action/Response Page
rat peripheral lymphocytes. Study title should include “Gasoline-DIPE Vapor

Condensate” Not necessary since the test substance is defined on the cover page. Accepted Suggest noting in the Summary section that the positive control fulfilled the requirements for a valid test. This was added to the Summary. Accepted p. 8

GFAP Study These studies appear to be pr operly conducted. Editorial comments from previous reviews regarding format, i.e. lack of study specifics, remain. Noted No changes were made in report format. Remain as prepared by Dr. O’Callaghan Vol IV Appendix AA

I concur with the overall conclusion that Gasoline-DIPE does not appear to result in brain gliosis as measured by enhanced GRAP regions. Noted

Micronucleus Study This study was properly conducted and scientifically sound. Positive and negative controls performed as expected and within historical range. Gasoline DIPE did not cause any

increases in the incidence of micronucleated mature erythrocytes and did not cau se any decreases in the proportion of immature erythrocytes. Thus, I concur with the report that Gasoline-DIPE Vapor Condensate does not cause an increase in frequency of micronuclei in immature erythrocytes. No evidence of bone marrow toxicity was

observed. Noted Vol IV Appendix X



### 211(b) Research Group Reviewer

General Comment

Summary p. 6, Vapor chamber concentration: Suggest that since

measured and nominal concentrations varied by ≤ 5%, that the variation should be considered “slight”, not

“somewhat”” and that the st atement concerning reasons for variations in calibration be deleted, especially since this statement does not appear in Results (p.47) Summary and related R&D clarified as suggested.

Accepted Summary p.6

Particle sizing, refer to Results (p.47). High dose value is 22% higher than control, mid dose is 14% higher. None of the other studies show these particle differences; controls were eq ual or higher than treated groups. Is more explanatio n needed in Results section and revision of statement in Summary? The only value of real interest for the comparison is the Total Mass Concentration

since that measures the amount of aerosol in the chambers. Since the TMC for all groups
is of the same order of magnitude (10 -3 ), no

difference is seen to discuss further. Accepted Sect 3.1 p.49

### 211(b) Toxicology Research Group Gasoline DIPE VC: A 13- Week Whole Body Toxicity Study in Rats with Neurotoxicity

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### Comments Action/Response Page
p. 7, Clinical Chemistry. A dd sentence addressing effects in

females since the absence of effects in males was specifically listed (see Results p.52, Sect 3.8.3). Revised sentence to address both sexes.

Accepted Summary p.7 Sect 3.8.3 p.53

Methods

p. 35, 2.15 Genotoxicity Evaluations : 1 st paragraph on Micronucleus study: In the sentence beginning “ BioReliance personnel removed the femurs..etc..., the second part “ which PRC personnel shipped to Eye Research Centre” is missing the word “shipped”. Please

add

p. 35, 2.16 Immunotoxicity p. 35, line 7: please spell sensiti zed with a z, rather than s.

p. 46 2.23 Protocol Deviations : Explain first deviation

more fully. How many measurements were not

collected in the “middle of th e study” – number of days

missed?? Corrected. Accepted

Corrected. Accepted

Clarified that ‘none’ of the measures made. Accepted Identified as week 6 Sect 2.15 p.36

Sect 2.16 p.36

Sect 2.23 p.47

Results and Discussion

p. 47 3.1 Chamber Monitoring : Add the variation value

( ≤ 5%) between nominal and an alytical concentration, since the percentage appears in the summary. p. 48 Particle Sizing: The high dose value (6.33 x 10 -3 ) is 22% higher than controls (5.17 x 10 -3 ). A variation of this size was not seen in any other gasoline + oxygenate study. Is this truly a “non substantial difference”? Is more explanation needed?

Restate somewhere in the Results that animals were treated for a minimum of 65 exposures over 13 weeks, perhaps at the end of the chamber monitoring paragraph [p. 47-48]. In earlier studies, peer reviewers have requested confirmation of the number of exposures, even though it is

stated in the methods.

p. 51, Table 10, 3.8.1 Hematology and Coagulation

(Appendix I): Suggest removal of modifiers such as “slight, very slight” from description of reticulocyte changes in male rats, and use of p ≤ 0.05 instead with percentages both here and in the Summary (p.6), unless there is an established definition to define “slight, very slight” in terms of % change.

What about statistically significant changes in platelets in males at wk 13 (p ≤ 0.05). Increases above control seem to plateau and drop slightly at high dose.

Does this warrant discussion? Added “<6%” to the text. Accepted

This difference is considered of no consequence since these are essentially all background values. Accepted

Upon review of prior reports, this information

was not added to the R&D and thus not added to this report. Accepted Information appears in Summary

Modifiers deleted and replaced with statistically significant and/or % change.

Accepted. Changes also recommended by Dr Goldsworthy [Checklist p.5]

Discussion of this inconsequential pattern

added to the section. Accepted Sect 3.1 p.48

Sect 3.1 particle sizing p.49

Summary p.5 first paragraph

Summary p.6 Sect 3.8.1 p.53

Sect 3.8.2 p.53

### 211(b) Toxicology Research Group Gasoline DIPE VC: A 13- Week Whole Body Toxicity Study in Rats with Neurotoxicity

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### Comments Action/Response Page

p. 52, Table 12, 3.8.3 Clinical Chemistry (Appendix K) This section, as written addr esses the absence of effects

in males. Subsequent sentences appear to refer to effects in females without stating that. Add “for females” in test substance-exposed groups in the 2 nd

sentence. Also, shouldn’t NA+, and K+ increases at wk

13 be mentioned? Section revised to address both sexes.

Sentence added to discuss the Na and K

values at terminal interval.

Accepted Sect 3.8.3 pp.53-54

### 211(b) Toxicology Research Group Gasoline DIPE VC: A 13- Week Whole Body Toxicity Study in Rats with Neurotoxicity

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### Comments Action/Response Page
Micronucleus Study Please add a table of male and female data separately as was done for the second draft of the Gasoline study.

Conclusions: Add “of either sex” to the conclusion that no significant increases ....were obser ved in rats “of either sex” treated with Gasoline DIPE vapor condensate. Table added. Accepted. Expanded Table 1

Added to the conclusion. Accepted Vol IV Appendix X p.19

p.15



### Sister Chromatid Exchange Study p. 11, Evaluation of Test Results, 1st paragraph: Revise

sentences addressing statistics to include the one-tailed Dunnett’s test and trend test requested by EPA.

p. 12 Results and Discussion: 4 th paragraph, 8 th sentence:

Revise sentence on use of Dunnett’s test to include onetail and trend analysis.

Table 6 p. 19: Air Control group Males AGT per group.

Check the value of 25 which appears to differ from a value of 26 in the interim table faxed to Gary Hoffman on 7-15-02 [Reproduced fax to me may have obscured the number]

Table 7 p. 20: 10,000mg/m 3 Females, MI per group.

Check the value of 5.3 which appears to differ from a value of 6.3 in the interim table faxed to Gary Hoffman on 7-15-02 [Reproduced fax to me may have obscured the number] Paragraph was revised. Accepted

Paragraph was revised. Accepted

The value of 25 is correct. Accepted

The value of 5.3 is correct. Accepted Vol IV Appendix Y p.11

p.12

p.19

p.20



### Immunotox Assay

Note: this section was om itted from the Checklist

Suggest that the last paragraph on p. 12 that describes the assay very clearly be moved to the Introduction (p. 10). It would fit very nicely as a second paragraph

Please provide a rationale for why only females were used in this assay. Peer revi ewers have requested this clarification in the other immunotoxicity studies.

The second table is missing table number Paragraph was not moved.

Sentences added indicating that females are more sensitive than males to hormonal [incl PFC in spleen] immuntox changes

Corrected Accepted Vol IV Appendix Z p.12 of 26

p.10 of 26

V.Introduction

Table 2



### GFAP Assay p. 8 Introduction : first full sentence at top of page – Change text as follows “Inhalation exposures to gasoline and gasoline plus each of the 6 fuel additives have been undertaken (replaces ‘will be” undertaken). Also, correct “with a two generation reproduction toxicity study that includes a neurotoxicity component” by adding ‘for gasoline and gasoline plus MTBE vapor condensates only.” Introduction revised Accepted Vol IV Appendix AA p.8

### 211(b) Toxicology Research Group Gasoline DIPE VC: A 13- Week Whole Body Toxicity Study in Rats with Neurotoxicity

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### Comments Action/Response Page
Although it has been decided by the Working group to maintain the GFAP reports in the current format, some general suggestions to make the report more consistent with other satellite studies are presented.

p. 14-16. V. Commentary A. Background information: This section belongs in the Introduction and can be integrated w ith the second paragraph beginning “ A universal cellular reaction......”

B. Typical Results: Figure 2 displays “typical GFAP assay values from different regions of rat brain.”

I assume these values are historical data from untreated control rats in Dr. O’Callaghan’s laboratory. Could the number of rats evaluated, strain, sex and age from which these data were collected be provided, perhaps in

a footnote? Do these vari ables affect the regional differences in GFAP values within a species? Title has been changed Accepted A. Backgroun d remains in this

section

“Historical” added \ Accepted

B. title is Critical Parameters D title is Typical Results Historical data visualized in Figure 2 has not been expanded in text. However text does indicate that “region to region differences of

untreated animals of a given species should

remain stable” pp.14-16

p.16 pp.16-17

p.17

Results and Conclusions : Please delete sentence concerning “ A recently developed fluorescence based GFAP ELISA assay for pituitary data....”. Although a ll of this information is of interest, it is appropriate in the discussion section of a publication, not in a study report for regulatory submission. These changes have already been made in

the 2 nd draft of the gasoline GFAP report. Deleted

Accepted p.17

Pituitary data: I am still not sure whether the pituitary data belongs in any of the oxygenate studies. Data is sketchy at best and the assay may not detect anything of value in this organ. Perhaps the pituitary data could be included in Table 7 individual data, but omitted from Tables 5 and 6, Mean GFAP levels in Male and Females, respectively. Only the high dose (3/5) and control (2/5) males and one control female pituitary data had any data. As had been seen in earlier GFAP studies on other gasoline + oxygenate blends, females seemed to provide much less data than males. Pituitary data deleted from Tables 5 & 6

Accepted Tables 5 and 6



### 211(b) Research Group QA/QC Reviewer

Audit Dates: May 3 –August 6, 2004

The draft final report (dated April 8, 2004 and Statistical Report of Behavioral Observations Noted.

### 211(b) Toxicology Research Group Gasoline DIPE VC: A 13- Week Whole Body Toxicity Study in Rats with Neurotoxicity

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### Comments Action/Response Page dated July 7, 2004) was audited to assure compliance with GLP regulations (Part 79.60) and to assure that the report represents the data collected. One hundred per cent of the report text

was audited and at least 10 percent of individual

data were verified.

1. 

### Quality Assurance Statement, Page 4, The Subcontractor Report and Study Data: To

what subcontractor does this refer? This has been deleted since the audits are all included in the QA Statements for the Satellite reports. OK

2. 

### Summary, Page 7, first paragraph, last sentence: This sentence refers only to the males having no exposure-related differences in clinical chemistry. What about the females? Sentence revised to discuss both sexes. OK



### 3. Methods, Section 2.2, Study Dates, pp. 12-13: This section of the report includes only a partial listing of significant study dates, primarily those for the main study and neuropathology animals. It is recommended that the section be expanded to include all relevant study schedule information including the following:

a. Date of animal receipt for immunotoxicity animals (also January 24, 2002).

b. Date of animal receipt for genotoxicity animals (March 4, 2002).

c. Terminal Sacrifice dates for immunotoxicity (March 14, 2002), genotoxicity (April 11, 2002) and GFAP (May 15, 2002) animals

d. 

### Exposure termination is reported as May 13-14, 2002; however,somesatellite groups were exposed through May 16, 2002. N ot revised to be consistent with prior reports in

t his test program. OK



### 4. Methods, Section 2.5.5, Age at Receipt and Initiation of Exposure, p. 18: The text correctly reports the age of animals at exposure initiation; however, the ages differ slightly from protocol requirements. For the main/recovery/neuropathology/GFAP groups, the protocol (Section 9.4.2) stated that animals would be approximately 8 weeks of age; these animals were approximately 9 weeks at exposure initiation. For the immunotoxicity groups, the protocol stated that animals would These minor differences are considered within the protocol requirements and do not require further notation in the report. The Study Director does sign in the raw data to accept animals for study. OK

### 211(b) Toxicology Research Group Gasoline DIPE VC: A 13- Week Whole Body Toxicity Study in Rats with Neurotoxicity

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### Comments Action/Response Page be approximately 6 weeks of age; these animals were actually 7 weeks of age at exposure initiation. These minor departures from protocol requirements should be addressed in the study data and report.

5. Methods, Section 2.5.7, Acclimation Period, p. 18: The text correctly states that animals were acclimated for up to three weeks; however, this differs slightly from protocol requirements. The protocol (section 9.5) specified an acclimation period of approximately two weeks. The actual length of acclimation was 19 days for main study/neuropathology/GFAP animals, 21 days for immunotoxicity animals and 11 days for genotoxicity animals. Therefore, some groups were acclimated for longer or shorter periods than required by protocol. These minor departures from protocol should be addr essed in the study data and report. These minor differences are considered within the protocol requirements and do not require further notation in the report. The Study Director does sign in the raw data to accept animals for study. OK



### 6. Methods, Section 2.9.7, Environmental Conditions, p. 21: Relative humidity in the animal housing room was occasionally above or below the protocol-required range of 30 to 70% (actual range was 25 to 76%, although daily averages were within protocol specifications). Excursions from protocol specifications should be addressed as protocol deviations in the study data and report, citing the duration of any excursions.

7. 

### Methods, Section 2.13, Neurobehavioral Studies, p. 28: The report text states that

neurobehavioral tes ting was conducted “on non-exposure days”. This same wording is used in the protocol (Section 11.5). This wording implies that neurobehavioral testing was scheduled for days on which exposures were not being conducted, when in actuality, animals scheduled for neurobehavioral testing were simply not exposed on those days when FOB and motor activity evaluations were performed (these days were regular exposure days). It is recommended that this be clarified in the report text. For future studies, some revision of the wording in the protocol should Based on protocol requirements and prior reports in this series of studies, we do not consider these excursions to be protocol

deviations.

Section clarified to avoid confusion to the

reader. OK

OK

### 211(b) Toxicology Research Group Gasoline DIPE VC: A 13- Week Whole Body Toxicity Study in Rats with Neurotoxicity

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### Comments Action/Response Page

be considered.

8. 

### Methods, Section 2.13.1, Functional Observational Battery Method, pp. 28-29: Some of the assessments made during FOB

evaluations are not included in the text’s description of the methods. For example, Handling Evaluations also included cage

removal and Reflex Assessments also included pinna reflex. It is recommended that the text be reviewed/revised as needed to accurately

reflect the FOB assessments conducted. Section revised as suggested. OK

9. 

### Methods, Section 2.15, Genotoxicity Evaluations, p. 34: Under SCE Evaluations, “heparin” is mistyped in line 8. Corrected. OK

10. 

### Methods, Section 2.15, Genotoxicity Evaluations, p. 35: Under Micronucleus Evaluations, line 10, the sentence is incomplete. It should indicate that the slides were shipped to Eye Research Center. Corrected. OK

11. 

### For Genotoxicity and Immunotoxicity evaluations, the protocol and report indicate that the Testing Facility was responsible for the GLP compliance status of the subcontractors (BioReliance and ImmunoTox, Inc.). Any facility and/or study specific inspections conducted by HLS to assess the GLP compliance status of the subcontractors should be included on the QA statement for this study. The inspections have been added as relevant to the QA Statements for each Satellite

report. Ok, but QA

Statements for

Gentox and GFAP reports

seem to be mixed up. GFAP necropsy

listed in Genotoxicity QA Statement

and vice versa.

12. 

### Methods, Section 2.17.2, Macroscopic Examinations, p. 36: A complete macroscopic examination was also performed on animals sacrificed in moribund condition during the study (i.e., GFAP animal 2070) in accordance with protocol requirements. The methods

section of the text should reflect that this animal was examined at necropsy. Added. Ok

13. 

### Methods, Section 2.17.4, Tissues Preserved and Examined Histopathologically, p. 37: The

second sentence states that slides of indicated tissues were prepared and examined microscopically for Main Study and Recovery animals. Microscopic evaluation of recovery animals was limited to gross lesions only. The text should be revised to clarify the scope of microscopic evaluations for the recovery Clarified. Ok

### 211(b) Toxicology Research Group Gasoline DIPE VC: A 13- Week Whole Body Toxicity Study in Rats with Neurotoxicity

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### Comments Action/Response Page

animals.

14. 

### Methods, Section 2.17.4, Screening for alpha 2u-globulin nephropathy, p. 37: The protocol (Section 12.6) states that kidney sections will be stained with Mallory Heidenhain stain if lesions indicative of nephropathy are seen in H & E stained sections. The additional staining was not performed for this study since these renal findings were expected and have been fully investigated in prior studies. However, a protocol amendment stating that the additional staining will not be performed was apparently not issued for this study. This departure from protocol requirements needs to be addressed in the study data and report. A protocol amendment #5 will be issued with

the 2 nd draft report to clarify that the special staining was not needed. Ok

15. 

### Methods, Section 2.17.4, Tissues Preserved and Examined Histopathologically, Table 1, pp. 37-38: Footnote “a” should appear next to “Bone marrow (femur)” in the list of tissues. Added Ok



### 16. Methods, Section 2.17.4, Tissues Preserved and Examined Histopathologically, Processing, p. 40: In line 13 of this text section, the abbreviation for Larynx, Ventral Seromucous Gland is given as V-SM-GLND. In the pathology tables and appendices, the

abbreviation used is V-SM-G. It is

recommended that the text be revised to consistently reflect the abbreviation used. Corrected sect ion 2.17.4. Ok

17. 

### Results, Section 3.2, Mortality, p. 49: Macroscopic examination of GFAP animal no. 2070 was performed at the time of moribund sacrifice. However, necropsy findings for this animal are not reported in either the main study or GFAP portions of the report. It is recommended that the report be revised to include necropsy observations for this animal. The macroscopic data added to the preface

page. Ok

18. 

### Results, Section 3.2, Mortality, p. 49: It is recommended that the report text be revised to specify the identity of the main study animal (i.e., no. 2060) used to replace no. 2070 (moribund sacrifice) for the purpose of GFAP evaluation. In addition, in-life data (clinical observations, body weights and food consumption measurem ents) for 2060 were collected in the main study protocol for the duration of the study and are reported in the Added replacement animal number to the

discussion.

Explanation for the data entries added to the preface page. Ok

ok

### 211(b) Toxicology Research Group Gasoline DIPE VC: A 13- Week Whole Body Toxicity Study in Rats with Neurotoxicity

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### Comments Action/Response Page tables and appendices for the main study animals. The report text should be revised to clearly indicate how data for the “GFAP replacement” animal were handled and reported.

19. 

### Results, Section 3.5, Body Weights, p. 50: The second sentence states that significant

increases were noted at weeks 9-11 in the 10,000 mg/m 3 

### females. The report tables for body weight change indicate that significant increases were present at weeks 9-12. The text should be revised accordingly. Corrected to weeks 9-12. Ok

20. 

### Results, Section 3.8.2, Coagulation, p.52: The text states that significant differences in coagulations parameters were either not in a treatment-related pattern or were minimal. It was noted that at week 4, APTT values for group II, III and IV males were significantly increased by 15-19% compared to controls. Is

this considered “minimal” difference or should this be further addressed in the report text? Added that these differences “were not similarly seen at the Terminal interval.” Ok

21. 

### Results, Section 3.8.3, Clinical Chemistry, p.

52: ALT should be included in the list of serum enzymes for which significant decreases

were noted. Added ALT to the discussion. Ok

22. 

### Results, Section 3.8.3, Clinical Chemistry, p. 52: Review of the clinical chemistry summary tables showed a number of significant differences with respect to mean electrolyte values (sodium, potassium chloride and/or calcium) in the treated groups compared to the controls at various intervals. For example, at termination, mean potassium levels in groups II, III and IV females were 15-25% higher than the respective control value. Should this, or any of the other significant differences in clinical chemistry parameters be further

discussed in the results section? Additional discussion added to the end of this

section. Ok

23. 

### Results, Section 3.10.2, Microscopic Pathology, p. 53: The text (line 2) states that “all tissues” from the control and high-dose exposure groups were examined following terminal sacrifice. In accordance with protocol requirements, there were some tissues collected and preserved at necropsy (e.g., eyes, mammary gland, muscle, pituitary, skin, etc.) Added. Ok

### 211(b) Toxicology Research Group Gasoline DIPE VC: A 13- Week Whole Body Toxicity Study in Rats with Neurotoxicity

Assessments and 4-Week In Vivo Genotoxicity and Immunotoxicity Assessments Checklist April 26, 2012

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### Comments Action/Response Page that were not examined microscopically. For clarity, it is recommended that the text be revised to indicate that “ all protocol-required tissues were examined following terminal

sacrifice”.

24. 

### Results, Section 3.10.2, Microscopic Pathology, p. 53: The text (lines 4-5) indicates

that no examinations were considered necessary for the recovery animals. The pathology reports indicate that gross lesions for the recovery animals were actually processed

and examined. The text should be revised to reflect the actual procedures for microscopic evaluation of the recovery animals. In addition, it was noted that the protocol did not specifically address the requirements for microscopic evaluation of tissues from recovery animals. It does not appear that the decision to examine gross lesions from these animals was addressed by protocol amendment and/or the decision documented in the study

records. It is recommended that documentation authorizing the examination of the lesions be included in the study file. The text is section 3.10.2 clarified that gross lesions were also examined for the Recovery

animals and clarifica tion also added to

Protocol Amendment #5. Ok

25. 

### Figure 2, Mean Body Weights, Males: The graphed value for Group IV at week 15 should be rechecked. The summary table reflects a mean of 554 grams for this interval. The graphed value appears to be incorrectly plotted as approximately 530 grams. Graph revised. Ok

26. 

### Appendix G and Table 8, Individual and Mean Motor Activity Values: The means and standard deviations reported in the table and appendix for Group IV males and females at Week 4 appear to be incorrectly calculated.

The calculated values for all intervals should

be rechecked and corr ected as needed. The statistical analysis report should be checked to determine if statistics were performed on the

correct mean values. The calculations have been checked and

found correct as is. Ok

27. 

### Appendix G, Individual Motor Activity Values: Incorrect motor activity values are reported for the following animals: Pretest, Group IV female 4573; Week 8, Group IV Female 4573; and Week 13, Group II The tables and appendices have been checked and corrected but the statistical report was not affected by these changes. Ok

### 211(b) Toxicology Research Group Gasoline DIPE VC: A 13- Week Whole Body Toxicity Study in Rats with Neurotoxicity

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### Comments Action/Response Page female 2567. In all three instances, the values reported for these animals appear to be a repeat of the results for the preceding animal. The report appendix should be

revised to reflect th e correct values for these animals and the corresponding summary tables and statistical analyses updated accordingly. It is recommended that this appendix be reviewed in its entirety to ensure that no other similar data transposition errors occurred during data

transfer.

28. 

### Appendix H and Table 9, Individual and Summary Functional Observational Battery Evaluations: Under Reflex Assessments, Visual Approach, the key included with Appendix H presents separate categories of 4 (jumps or turns abruptly to avoid) and 5 (attacks and/or bites. In the summary table, these descriptions are co mbined into a single category (jumps or turns abruptly to avoid or attacks and/or bites). The table and appendix should consistently categorize the responses for this parameter and s hould also be consistent with the data and SOP for this procedure. The appendix has been revised to be

consistent with the table and the data. Ok

29. 

### Table 9, Summary of Functional Observational Battery Evaluations: For Week 13, Group IV Females, the reported incidences for Pinna Reflex are incorrectly reported. Appendix H indicates that nine animals in this group exhibited scores of 2 (animal shakes head) and one animal exhibited a score of 1 (ear flattens against head). The summary table incorrectly reports incidences of 0 and 10 for scores 1 and 2, respectively. The statistical analysis report should be checked, as appropriate, to verify the accuracy of the data used for statistical analysis. The summary table has been corrected but the statistical report was unaffected. Ok

30. 

### Table 10, Mean Hematology Values: For the Terminal interval, “Huntingdon” is mistyped in

the table headers. Corrected. Ok

31. 

### Table 12, Mean Clinical Chemistry Values: The summary tables for clinical chemistry parameters do not report the means and standard deviations for gamma-glutamyl Table reprinted to present the GGT data. Ok

### 211(b) Toxicology Research Group Gasoline DIPE VC: A 13- Week Whole Body Toxicity Study in Rats with Neurotoxicity

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### Comments Action/Response Page transferase at any interval. The tables should

be revised to include GGT data.

32. 

### Appendix I, Individual Hematology

Values: Manual differential counts were performed and reported for several animals, including: Group I male 1063, Week 4; Group I male 1068 and Group IV male 4062, Termination; Group I female 1568, Group II female 2559 and Group IV females 4563 and 4569, Termination; and Group I male 1057, Recovery. It is

recommended that these animals be footnoted in the appendix to indicate that manual readings are reported. In addition, the keys for the Hematology appendix and table should reflect that “absolute large

unstained cells” are not evaluated in manual differential counts and are reported

as 0 in these instances. The preface page has been denoted for these

details. Ok

33. 

### Appendix I, Individual Hematology

Values: Please recheck the calculated values for absolute neutrophils and absolute lymphocytes for Group IV female

4563 at Termination. This animal had a manual differential count, and based on the reported counts of 20 and 77 for neutrophils and lymphocytes, respectively and a total WBC value of 10.81, my

calculations for absolute values were 2.16 and 8.32, respectively. Values of 2.06 and 7.93 are reported in the appendix. This may affect the summary tables and

statistics. The data for both samples were corrected in ClinAxys and on the Hematology manual

differential form. Ok

34. 

### Appendix K, Individual Clinical Chemistry Values: The procedure for reporting bile

acids values above 200 μ mol/L appears to be inconsistent. In some instances, results above 200 are reported as “>200” in the appendix and are not included in the calculation of group means and standard deviations (see for example, Week 4, Group I female 1565, Group II female The samples that were diluted because of instrument and did achieve a quantifiable

value are true and correct results.

Those samples that were reported as >200

even after dilution did not achieve a true quantifiable value. Thus they were reported as >200. This does confirm that the original samples were elevated and reported correctly.

In addition to this, there was evidence in the Ok. (Even though values are reported individually as >200, they are

not included in a mean. They

are known to be

elevated so the

means for these

groups seem

### 211(b) Toxicology Research Group Gasoline DIPE VC: A 13- Week Whole Body Toxicity Study in Rats with Neurotoxicity

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### Comments Action/Response Page 2557, Group III female 3552 and Group IV female 4568). In other instances, results above 200 are reported as the actual value

obtained and are incl uded in the group means and standard deviations [see for example, Week 4, Group II male 2056, (value of 302.8) and Group III male 3055 (value of 221.4) and Recovery, Group 4 female 4559 (value of 210.8)]. Please clarify why some values above 200 are quantified and reporte d while others are

not. Should all values above 200 be excluded from group mean calculations? Summary tables and statistics may need to be revised if changes are made to these

data. data that many samples were low in volume which may have contributed to the inability to further dilute samples a second time. inaccurate.)

35. 

### Appendix M, Individual Gross and Microscopic Observations and Study Data, Histotechnology Records: For Group IV males 4067 and 4069 (terminal sacrifice), the microscopic examination indicates poor

fixation of the testes. Notations in the Histotechnology records i ndicate that it did not appear that testes from these animals

were fixed in modified Davidson’s solution at necropsy, as require d by protocol. This possible departure from protocol specifications should be addressed in the study data and report. Protocol deviation signed by the SD and added to the data and the report. Ok

36. 

### Appendix M, Individual Gross and Microscopic Observations and Study Data, Histotechnology Records: The histotechnology record for animal 3562 documents a post-fixation observation for this animal (lungs, all lobes discolored). This observation was apparently not entered into the pathology program as it does not appear in Appendix M of the draft report. This observation should be entered for reporting purposes and microscopic correlation (or lack thereof) provided for

this lesion. The post-fixation observations have been entered into Xybion an d correlated by the pathologist. Ok

### 211(b) Toxicology Research Group Gasoline DIPE VC: A 13- Week Whole Body Toxicity Study in Rats with Neurotoxicity

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### Comments Action/Response Page

37. 

### Appendix N, Pressure/Vacuum Gauges and Regulators: I believe these are R07 and not PN 219RGKA. Please verify. Corrected. Ok

38. 

### Appendix O, Density of DIPE: The density used was 0.64 per the density worksheet. Please clarify. Corrected. Ok

39. 

### Appendix S, Analytical Report: The validation report for DIPE contains a typical chromatogram for Study 6125 (BGVC). Shouldn’t it include one of

DIPE? A typical chromatogram was used because it was difficult to insert specific chromatograms in the validation reports for these studies. Also, due to long runtimes, the chromatograms are not very clear when compressed onto one page. Ok

40. 

### Appendix S, Analytical Report, Page 5: Could an assessment of the stability and composition of the test material be given? All samples were analyzed on the day they were received. Stability analysis was not required.

Sections 2.4.6 states that, “The test substance stability was tested concurrently with the study and the results (see Appendix S)

showed it to be stable for the duration of the
study 

### .” Ok- Comment

was intended for the report to

assess the test

material stability and composition through the length of the study.

41. 

### Appendix S, Analytical Report, Figure 2: This does not appear to be the chromatogram from this study 2001A sample. Please clarify. Figure 2 is Sample 2001, injection 004-007 from this study. ok

42. 

### Appendix T, Neurobehavioral Positive

Control Data: The titl e pages to these two studies are missing. Th ere is no indication of the study number. It appears to be part of the 00-6130 Study, which it is not. Its study number is 00-14500, right? Is the Motor Activity portion part of that study or a different one? Please clarify that this is not part of the API study. The FOB and MA reports have been replaced. ok

43. 

### Study Data, Pre-Study Room Preparation Form, Plant Maintenance Section: The section that documents adjustment of water pressure is left blank on the form. This data recording omission should be

addressed. Corrected ok

44. 

### Study Data, Authorization to Order

Animals: The authorization form for ordering genotoxicology animals indicates There is no notation in the data that this was approved by the Study Director at time of animal receipt however the Study Director did approve the weights of the animals at the ok

### 211(b) Toxicology Research Group Gasoline DIPE VC: A 13- Week Whole Body Toxicity Study in Rats with Neurotoxicity

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### Comments Action/Response Page that these animals were to weigh 75 - 125 grams at receipt. The weight list from the animal supplier indicates that the animals weighed approximately 50 –75 grams prior to shipment. Was this lower weight range authorized/approved at the time of receipt? What procedures are followed to assure that animals received meet the accepted weight requirements? time of randomization into test groups to start the study exposures.

45. 

### Study Data, Bleeding List for Week 13, May 14, 2002: A comment on the bleeding

list for animal no. 2060 references the transfer sheet for details concerning the lack of blood samples for this animal. The transfer sheet does not provide any explanation, but the fact that this animal was used as a replacement for GFAP evaluation is clearly documented in other study documentation (memos, etc.). For clarity, it is recommended that the comment on the bleeding list be changed to reference the study memos rather than the

transfer sheet. Comment added to the bleeding list to refer

to memo dated 5March03. ok

46. 

### Study Data, Bleeding List for Coagulation Samples, Week 13, May 14, 2002: The label for documenting centrifuge parameters documents two “in” times, but

the second “out” time was not recorded. This data recording omission should be

addressed on the form. SOP deviation documented in the raw data. ok

47. 

### Study Data, Bleeding List for Clinical Chemistry Samples, Week 4, March 13, 2002: The label for documenting centrifuge parameters for centrifuge CP037 documents two “in” times, but the second

“out” time was not recorded. This data recording omission should be addressed on

the form. SOP deviation documented in the raw data. ok



### 48. Study Data, Hitachi 717 Worksheet for Week 4, March 13, 2002: Footnote “14”

on the worksheet indicates that due to insufficient sample volume, some protocol The footnote has been added to the Hitachi raw data print out. ok

### 211(b) Toxicology Research Group Gasoline DIPE VC: A 13- Week Whole Body Toxicity Study in Rats with Neurotoxicity

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### Comments Action/Response Page specified tests were not completed for

some animals and the results would be reported as QNS. However, footnote “14” does not appear to be noted in the Data Monitor Reports (clinical chemistry raw data) for correlation purposes. For completeness and clarity, these data recording omissions should be addressed in the Data Monitor Reports.

49. 

### There are sample transfer records for large cylinders returned to the sponsor. These

must have been returned to Chevron. Also, on 3/15/02 there was 1 large cylinder

sent. Was this one full? There is a memo indicating DIPE container number 6 was

received 8/5/02. It a ppears that this should have only been container number 5 and the

one received 9/3/02 should have been 6. That is correct – all return shipments were to Chevron from where the cylinders originated.

This cylinder was empty and note added to the data in Pharmacy file.

Memo corrected to reflect that the cylinder number was actually number 5. There is no record of a receipt on 3Sept02 of a cylinder containing G/DIPE. This was probably another test substance in 211b testing. Ok

Ok

ok

50. 

### It appears that Large Cylinders number 1,2, and 3 were used for this study. All of 1

and 2 and about 20% of number 3. This indicates about 500,000 ml DIPE used. If one looks at the amount actually measured, however, there was substantially less used (approximately 5090 ml/day for 70 days) at 356,300 ml. Is there a logical explanation for this difference or are my calculations

incorrect? The ~500,000 mL is the amount of G/DIPE dispensed, not what was actually used as it was not possible to completely empty the small cylinders when used on test. Some material remained in the small cylinders (not used) which would account for some of the difference. Additionally, some of the small cylinders filled for use on 00-6130 were not used on this study but were instead used on study 00-4205. The amount of G/DIPE used in 00-4205 for those cylinders is not

accounted for in th ese calculations. ok

51. 

### The inventory for the large cylinder used to fill 1A1-1T1 appeared incorrect. The cumulative amounts were incorrect, although the sum was correct. Large cylinder #1 Test Material Dispensing Log cumulative weight corrected previously on 09 Sep 04 on separate Test Material Dispensing Log form. Notes added to both logs to link corrected sheet with original. ok

52. 

### In Vivo-In Vitro Rat Peripheral Lymphocyte Sister Chromatid Exchange Assay Draft Report: The Statement of Compliance now does not contain the statement about the positive control

articles. Is this intentional? These statements have been re-inserted into this page. ok

53. 

### In Vivo-In Vitro Rat Peripheral Lymphocyte Sister Chromatid Exchange This line defined as “Experimental Start Date

for SCE” and therefore the date for the SCE ok

### 211(b) Toxicology Research Group Gasoline DIPE VC: A 13- Week Whole Body Toxicity Study in Rats with Neurotoxicity

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### Comments Action/Response Page Assay Draft Report, Page 6: The Experimental Start and Completion Dates

should still be the same as in-life phase, I believe. The experimental start date is defined as the date the test system begins receiving the test material. This should be

3/15/02. sampling is relevant and included.

54. 

### In Vivo-In Vitro Rat Peripheral Lymphocyte Sister Chromatid Exchange Assay Draft Report, Table 6: The Range of SCEs per animal for animal number 2071 should be 4-10; animal 2072 should be 4-12; for 2074 shoul d be 5-9; and for

animal 3072 should be 4-10. These ranges have been corrected. ok

55. 

### In Vivo-In Vitro Rat Peripheral Lymphocyte Sister Chromatid Exchange Assay Draft Report, Table 7, Range of SCEs per animal: For animal 4592 should

be 3-9. The range has been corrected. ok



### 56. Immunological Evaluation of Gasoline DIPE Vapor in Female SD Rats Using the Plaque Forming Cell Assay Report, Page 13 of 24: The terminal body weight of the vehicle-exposed rats was 251.6 with a standard deviation of 13.5. The 4.3 figure

is a standard error of the mean. ImmunoTox, Inc. presented the data in the report as means with standard errors, not

standard deviations. ok

57. 

### Immunological Evaluation of Gasoline DIPE Vapor in Female SD Rats Using the Plaque Forming Cell Assay Report, Table

2: The number “2” needs to be added to

the table. Added. Not Added

58. 

### GFAP Levels in Specific Rat Brain Areas Following a 13 Week Whole Body Inhalation Exposure to DIPE Report, Page 8: Please verify the terminal sacrifice date. The exposure records indicate exposure on

the 15 th 

### of May. Sacrifice date 15 May 2002 added and other earlier study dates added to report ok

59. 

### GFAP Levels in Specific Rat Brain Areas Following a 13 Week Whole Body Inhalation Exposure to DIPE Report, Table 6, Hypothalamus, Group 1: The mean Corrected to 6.28. Ok

### 211(b) Toxicology Research Group Gasoline DIPE VC: A 13- Week Whole Body Toxicity Study in Rats with Neurotoxicity

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25 4/26/2012 

### Comments Action/Response Page

should be 6.28. The mean for the Thalamus for the Group 4 females should be 1.75. Also the there is only one value for the Group 1 female pituitary so a SEM should not be given. The Group 4 pituitary

value is listed as 0.21 in the statistical print-out. Where would it have come

from? No, correct as 1.76.

Pituitary data removed. Ok

ok

60. 

### GFAP Levels in Specific Rat Brain Areas Following a 13 Week Whole Body Inhalation Exposure to DIPE Report: The last female listed on the page should be 4577 not 2568. The data reported there is for 4577. The data reported for Group 2

female 2568 is correct. Corrected to 4577 ok

61. 

### GFAP Levels in Specific Rat Brain Areas Following a 13 Week Whole Body Inhalation Exposure to DIPE Report:

Please check the Striatum value for animal number 1080. The GFAP value is 5.472,

not 12.517. Should be 1.148 not 12.517 and corrected. ok

62. 

### GFAP Levels in Specific Rat Brain Areas Following a 13 Week Whole Body Inhalation Exposure to DIPE Report: Please check the Hippocampus value for

animal number 4578 as the BCA value may have been incorrect (9.465 vs. 9.805). 9.805 is correct value ok

63. 

### GFAP Levels in Specific Rat Brain Areas Following a 13 Week Whole Body Inhalation Exposure to DIPE Report: Please label Figures 1 an d 2 with the origin

of these data. Added information to Figure Legends ok

64. 

### GFAP Data: At the 100 dilution for the Striatum, samples 4, 8, 24, and 35 were repeated, but I could not find the repeat

values. Done on 6/11/02 with other repeated samples & Pituitary samples. ok

65. 

### GFAP Assay Data: There is a BSA Standard Stock solution mixing date of 10/4/02. Since the assay was done later in May of 2004, it probably should have been

10/4/01. Agreed and corrected in data. ok

### 211(b) Toxicology Research Group Gasoline DIPE VC: A 13- Week Whole Body Toxicity Study in Rats with Neurotoxicity

Assessments and 4-Week In Vivo Genotoxicity and Immunotoxicity Assessments Checklist April 26, 2012

26 4/26/2012  Additional items noted – The Micronucleus report appears to be from an earlier version of this report. Page 1066 of 1439 contains several ? marks for dates, lot numbers, and purity of the test

material. There are a few different tables and appendices as well.

 Table 7 is actually the Statistics Report from ExxonMobil. It needs a cover page to indicate this (was in draft 1although report was not). Please verify that QA has reviewed this report.

 The QA Statement for CD C (GFAP) is blank.

 Experimental Termination and Study Completion dates will need to be added to report.

 

### Immunological Evaluation of Gasoline DIPE Vapor in Female SD Rats Using the Plaque Forming Cell Assay Report, Table 2: The number “2” needs to be added

to the table.