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 

1 4/26/2012 

Comments Action/Response Page 

EPA 

Overall, the NHEERL reviewers find that the methods 

employed were in accordance 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 saying 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 statement 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 

Assessments and 4-Week In Vivo Genotoxicity and 

Immunotoxicity Assessments 

Checklist April 26, 2012 

2 4/26/2012 

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 example, 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 indicated 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 

Assessments and 4-Week In Vivo Genotoxicity and 

Immunotoxicity Assessments 

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3 4/26/2012 

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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. Also unclear is the basis for the 

statement that there is an increase in activity in the females 

at mid-exposure during the 4th 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 statistically 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 numbers 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 

Assessments and 4-Week In Vivo Genotoxicity and 

Immunotoxicity Assessments 

Checklist April 26, 2012 

4 4/26/2012 

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 levels 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 study 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 procedures 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 proportions. 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 observed in the 20,000 mg/M3

 exposed 

animals; these observations abated 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 

Assessments and 4-Week In Vivo Genotoxicity and 

Immunotoxicity Assessments 

Checklist April 26, 2012 

5 4/26/2012 

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 , 2nd 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 and 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 “statistical (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 chemistry 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 

Assessments and 4-Week In Vivo Genotoxicity and 

Immunotoxicity Assessments 

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6 4/26/2012 

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 these defects adverse since no 

histopathology changes were noted and decreases were 

relatively small. 

No exposure-related differences 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/M3

 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/M3

 ) 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 study results. This conclusion, 

as stated on p.9 and p.17, could note at what exposure level 

the decrease occurred, and state a NOAEL of 10,000 mg/M3

 

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 

Assessments and 4-Week In Vivo Genotoxicity and 

Immunotoxicity Assessments 

Checklist April 26, 2012 

7 4/26/2012 

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 properly 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 cause 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 statement 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 equal or higher than treated 

groups. Is more explanation 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 

Assessments and 4-Week In Vivo Genotoxicity and 

Immunotoxicity Assessments 

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8 4/26/2012 

Comments Action/Response Page 

p. 7, Clinical Chemistry. Add 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: 1st 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 sensitized 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 the 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 analytical 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 

Assessments and 4-Week In Vivo Genotoxicity and 

Immunotoxicity Assessments 

Checklist April 26, 2012 

9 4/26/2012 

Comments Action/Response Page 

p. 52, Table 12, 3.8.3 Clinical Chemistry (Appendix K) 

 This section, as written addresses 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 2nd

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 

Assessments and 4-Week In Vivo Genotoxicity and 

Immunotoxicity Assessments 

Checklist April 26, 2012 

10 4/26/2012 

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 observed 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: 4th paragraph, 8th 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/m3

 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 omitted 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 reviewers 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: 

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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 with 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 variables affect the regional 

differences in GFAP values within a species? 

Title has been changed 

Accepted A. Background 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 all 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 2nd 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: 

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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 percent 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. 

Not revised to be consistent with prior reports in 

this 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: 

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Assessments and 4-Week In Vivo Genotoxicity and 

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Checklist April 26, 2012 

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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 addressed 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 testing 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: 

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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: 

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Assessments and 4-Week In Vivo Genotoxicity and 

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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 2nd 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 section 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 measurements) 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: 

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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/m3

 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: 

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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 clarification 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 corrected 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: 

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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 the 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 combined 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 should 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: 

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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 μmol/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: 

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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 included 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 reported 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 indicate that it did 

not appear that testes from these animals 

were fixed in modified Davidson’s solution 

at necropsy, as required 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 and correlated by the 

pathologist. 

Ok 
211(b) Toxicology Research Group Gasoline DIPE VC: 

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Assessments and 4-Week In Vivo Genotoxicity and 

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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 title pages to these two 

studies are missing. There 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: 

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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 

Assessments and 4-Week In Vivo Genotoxicity and 

Immunotoxicity Assessments 

Checklist April 26, 2012 

23 4/26/2012 

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 appears 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 these 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 

Assessments and 4-Week In Vivo Genotoxicity and 

Immunotoxicity Assessments 

Checklist April 26, 2012 

24 4/26/2012 

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 should 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 15th 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 

Assessments and 4-Week In Vivo Genotoxicity and 

Immunotoxicity Assessments 

Checklist April 26, 2012 

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 and 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 CDC (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.