211(b) Toxicology Research Group Gasoline TBA VC:

13- Week Subchronic Toxicity and FAB/MA Neurotoxicity 

Reviewer Checklist 

1 5/25/2012

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 concur that no adverse effects of 

treatment were observed in the genotoxicity, 

immunotoxicity or glial fibrillary acidic protein 

assessments. We concur also that increase motor 

activity, increased anogenital staining, and increased 

blood cholesterol concentrations observed at the top 

dose level should be considered as adverse health

outcomes.

Noted.

Our review identified an error in regard to reported 

results of the landing foot splay measure in the 

functional observational battery. The report states that 

mid dose level was different from controls at the 8 

week time point, however, the statistically significant 

dose-time interaction involved the low dose level and 

not the mid dose level (Volume I of III, Summary of 

FOB and Motor Activity Analyses, Table II) This 

error occurs in the test of the statistical report and is 

carried over to the rest of the report. Also, the 13 

week time point is referred to as week 12 in the same 

table.

The Statistical Report by Mark Nicolich 

rewritten based on new data and 

interpretation and the main report 

Summary and R&D updated as needed.

Accepted. 

Summary p. 5

Results p. 52

Statistics 

report on p. 

123

In addition, we question the interpretation of the foot 

splay data provided in the report. The statistical 

significant interaction is interpreted as the control 

group being “higher than expected” at 8 weeks. 

However, it is clear that the mean foot splay value of 

the control group at 8 weeks is essentially identical to 

that at 4 weeks, and therefore should not be considered 

as higher than expected. In contrast, foot splay values 

of the low dose group decreased markedly from 4 to 8 

weeks, which apparently was the key factor giving rise 

to the statistically significant interaction. Never-theless, the decreased foot splay observation was not dose 

related and peripheral neuropathies typically result in 

an increased and not a decreased landing foot splay 

measure. For these reasons, we do not suggest that 

this outcome is toxicologically significant.

The Statistical Report by Mark Nicolich 

rewritten based on new data and 

interpretation and the main report 

Summary and R&D updated as needed.

Accepted

Summary p. 5

Results p. 52

Statistics 

report on p. 

123

Peer Reviewer – Schlesinger

General Comments

The investigators should indicate the groups as either 

I, II, III or IV or 1,2,3,4

but should not use both types of numbers. There is a 

lack of consistency in the report.

This cannot be changed within the 

limitations of the computer systems in 

use for this report

Accepted.

There does not seem to be a uniform statistical "p" Each statistical routine has its own 
211(b) Toxicology Research Group Gasoline TBA VC:

13- Week Subchronic Toxicity and FAB/MA Neurotoxicity 

Reviewer Checklist 

2 5/25/2012

Comments Action/Response Page

level for all of the auxiliary tests. This

should be addressed.

predetermined “p” level(s) and cannot be 

changed.

Accepted

Main Report

p. 42. Section 2.20.1. The investigators could have 

used a two way ANOV A that would

have allowed evaluation of both time intervals. They 

should indicate why this approach

was not used. Furthermore, the use of both 0.1 and 0.5 

levels of significance needs to be

addressed. Generally, one or the other is employed and 

there is not necessarily

consistency throughout the various aspects ofthe study 

in this regard.

This was the standard statistical package 

at this laboratory at the time of this study 

and cannot be changed.

Accepted

p. 48. Section 3.1 The investigators need to indicate 

why there may have been " slight

inaccuracies" in the calibration of the chamber 

airflows or the IR monitor. Monitor

calibration especially should be highly accurate if 

properly performed. The fact that the

control chamber contained particles suggests that there 

was no particle filtration of

incoming air. If this is true, why were filters not used.

This section and the related section in the 

Summary revised to reflect the 

acceptability of the small differences.

Accepted

p. 48

p. 50. Section 3.5. The mean BW in females in the 

figure seems to be decreased by

exposure compared to control, especially in Group IV. 

This is not consistent with the

description provided in this section in the first 

sentence. In addition, what is the basis to

indicate that the decrease in body weight gain was 

only transient?

This section and the related section in the 

Summary clarified that “no adverse” 

effect on body weights was seen and 

„transient‟ removed.

Accepted

p. 51

p. 49. Section 3.3.2. It is noted that an exposure related 

increase in the frequency of

yellow ano-genital staining was seen in the high level 

exposed females and the other test

animals. Which other animals are the investigators 

referring to since this effect does not

seem to be consistent across all groups.

Section clarified that only compared to 

control females.

Accepted

p. 50

p. 52. Section 3.8.1. The description of effects as 

noted in this section needs to be

reevaluated. The data sets seems to indicate a number 

of what appear to be treatment

related effects. A test substance may produce an effect 

on one endpoint without affecting

others, such as the immune system or histopathology. 

Thus, effects should not be

discounted merely because other endpoints were not 

affected.

Upon further review by our Clinical 

Pathologist, we have decided that the 

original conclusion of „no effect‟ are still 

valid and therefore the text not revised.

Accepted

p. 53
211(b) Toxicology Research Group Gasoline TBA VC:

13- Week Subchronic Toxicity and FAB/MA Neurotoxicity 

Reviewer Checklist 

3 5/25/2012

Comments Action/Response Page

p. 53. Section 3.8.3. What is the basis for the 

statement that serum enzyme decreases

" ... are generally not considered indicative of test 

substance-induced toxicity ... ?

This is based on tables in Gad et al, 

“Statistics and Experimental Design for 

Toxicologists”, 1988.

Accepted

p. 54

Opthalmologic Examination Report; 

No concerns.

Noted.

Peer Reviewer – Goldsworthy

 The potential inhalation toxicity of G-TBA 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; all reviews presented below). The report was 

clearly written and comprehensive. The design of the 

study was appropriate and sound and the study was 

properly conducted. Environmental conditions 

(temperature, humidity, noise level, illumination) were 

appropriate. The deviations noted had no impact on 

the overall study conclusions or data integrity; the lack 

of the Mallory Heidenhain staining to confirm the 

presence of alpha 2u-globulin nephropathy does not 

permit the report to definitely state this expected 

finding - for consistency and to place this sole major 

finding within proper context, I recommend staining 

with Mallory‟s on at least some high dose animals.

Noted and protocol amendment#5 has 

been drafted to clarify the sponsor 

decision not to do special staining for this 

study.

 Exposure techniques and procedures were adequate 

to test the potential toxicity of G-TBA. Exposure 

levels of test substance were relatively close to target 

levels. Animals were exposed to all major components 

of G-TBA in their expected proportions. Week to 

week data consistency was noted, indicating stability 

of test substance and proper techniques.

 Experimental data revealed no effect of G-TBA on 

survival, feed consumption or gross abnormalities at 

necropsy. Yellow ano-genital staining was observed in 

high exposed female animals; these observations 

abated following the 4 week recovery period. No 

exposure related changes were noted in absolute body 

weights or in final body weight changes; a transient 

decrease in body weight gain was observed for the 

first 4 weeks of exposure in high dosed female rats. 

No exposure-related effect was observed with 

opthalmoscopic assessments. 

 G-TBA exposure did not appear to be associated 

with FOB measurement count changes; foot splay data 

indicated a potential change that was deemed not 

Noted.
211(b) Toxicology Research Group Gasoline TBA VC:

13- Week Subchronic Toxicity and FAB/MA Neurotoxicity 

Reviewer Checklist 

4 5/25/2012

Comments Action/Response Page

exposure related. An increase in motor activity during 

exposure and recovery periods were seen in the high 

exposure group relative to the control group. The 

revised statistical procedures undertaken (Protocol 

Amendments 1 and 2) appear appropriate to assess the 

responses observed. 

 Effects of G-TBA exposure were noted in 

hematology parameters and coagulation values in 

Table 10; report notes and suggests that these slight 

differences were most likely attributable to normal 

variation and supported by lack of evidence of 

immunotoxicity or histopathological changes. 

However, the data demonstrating decreased reductions 

in lymphocytes counts and decreased basophil counts 

are (a) different in both male and female exposed 

animals, (b) at both the 4 and 13 week time points, and 

(c) often seen in the mid and high exposed groups. 

Furthermore, these changes were often statistically 

different, and when they did not reach statistical 

significance level, they were still decreased relative to 

control values. I would consider these changes to be 

attributable to G-TBA exposure and thus Report text 

(Summary, Conclusions, and Results - page 52) may 

be modified to reflect this.

Upon further review by our Clinical 

Pathologist, we have decided that the 

original conclusion of „no effect‟ are still 

valid and therefore the text not revised.

Accepted

p. 53

Serum cholesterol values were increased in the mid 

and high exposed male rats; this difference abated by 

the end of the recovery period. It is interesting that this 

change was only noted in male rats. No other 

significant alterations in clinical chemistry were 

attributed to G-TBA exposure.

Noted.

 Absolute and/or relative to body or brain weight 

differences in kidney weights were observed in mid 

and high exposed male rats. These increases were 

expected, consistent with microscopic findings, and 

most likely due to hydrocarbon-induced nephropathy. 

No other exposure related differences in organ weight 

were noted.

Noted.

 Microscopic findings were present in the kidneys of 

high dose male rats. These renal findings were 

consistent with hydrocarbon-induced nephropathy 

(even though the presence of alpha 2u-globulin was 

not confirmed by Mallory staining). The kidneys of 

the mid level exposure group in male rats were not 

examined microscopically and thus it could be 

determined if nephropathy was also present at the 

10,000 mg/m3exposure group. This may be expected 

since renal weights were also increased at the mid 

Clarified in the Summary and R&D and 

Conclusion that “(the 2000 and 10000 

mg/m3 exposed animals were not 

examined)”

Accepted

Summary p. 7

Results p. 55

Conclusions p. 

57
211(b) Toxicology Research Group Gasoline TBA VC:

13- Week Subchronic Toxicity and FAB/MA Neurotoxicity 

Reviewer Checklist 

5 5/25/2012

Comments Action/Response Page

exposure level. The text is written inappropriately 

regarding the induction of hydrocarbon induced 

nephropathy in that this finding is suggested to occur 

only in the high exposure group of male animals. Text 

should be reworded (Results, Conclusions, Summary) 

to more accurately reflect what is known and what is 

not (e.g. only see high dose effect because only 

examined high dose).

 Summary: In addition to the above comment on 

nephropathy, Conclusion (as well as Summary and 

Results - page 56) should note the increased renal 

weights at the mid and high exposure groups in male 

rats. I concur that the 2000 mg/m3 exposure level be 

considered the NOAEL.

Clarified the conclusion in the Summary 

and the R&D for the kidneys weighs.

Accepted

Summary p. 7

Results p. 54

Conclusions p. 

57

Editorial: The Table on Page 14, Section 2.3 

Experimental Outline, lists 10 male rats in the Control 

Recovery Microscopic Pathology - Recovery Group; I 

believe this should be 9, as seen in the other recovery 

group listings.

Corrected.

Accepted

p. 15

211(b) Research Group Reviewer

Summary

p. 6, Exposure Concentrations: Suggest that the 

sentence discussing slight inaccuracies in 

calibration be deleted since the variations between 

analytical and nominal concentrations were less 

than 0.1% at 2000 and 1000mg/m3and <5.0% at 

20000mg/m3, and both values, especially 

analytical, were close to the target concentrations.

Deleted from the Summary and R&D

Accepted

p. 6

Body weight: Suggest adding the word “significant” 

in the sentence addressing the absence of exposure 

related body wt changes since high dose females 

had some transient wt gain decrements during wk 

0-4. Sentence can then read “There were no 

significant exposure-related differences in body 

weights or feed consumption, etc.....”

Per prior comment corrected to no 

„adverse‟ differences.

Accepted

p. 6

Hematology: Changes in some hematology 

parameters have raised concerns from both peer 

reviewers and should be noted here and described 

fully in the report text.

Upon further review by our Clinical 

Pathologist, we have decided that the 

original conclusion of „no effect‟ are still 

valid and therefore the text not revised.

Accepted

p. 6

p.7, Organ Weights and Histopathology: Suggest 

reiterating that only kidneys of high dose and 

control animals were examined histologically 

Added both items to the Summary.

Accepted

p. 7
211(b) Toxicology Research Group Gasoline TBA VC:

13- Week Subchronic Toxicity and FAB/MA Neurotoxicity 

Reviewer Checklist 

6 5/25/2012

Comments Action/Response Page

[although this was indicated earlier in the 

Summary] and that the presence of alpha 2-

microglobulin is likely, on the basis of past 

experience in other studies in this series but was 

not verified by Mallory-Heidenhain stain (entry 

requested by Dr. Goldsworthy). 

2.3 Experimental Outline (p.14): Correct entry 

for Microscopic Pathology, Recovery Males to 

9. Current entry of 10 male rats is incorrect.

Corrected.

Accepted

p. 15

2.23 Protocol Deviations (p.46): Item 4. I thought 

that the Work Group had agreed not to use special 

stains to confirm alpha 2-microglobulin after 

doing the confirming work with baseline gasoline 

and BG+MTBE. Why is this a deviation from the 

protocol in the final report? Shouldn‟t this have 

been listed as a protocol ammendment? Section 

2.17.4 of Methods specifically states that MalloryHeidenhain stain would not be used. Also the 

DIPE report [at least] does not list this as a 

protocol deviation and special staining was not 

used on kidney sections in that report. Suggest 

this entry be deleted for consistency with other 

studies. Please check.

Deleted from the M&M and have drafted 

protocol amendment#5 to correct the 

protocol.

Accepted

p. 47

3.1 Chamber Monitoring: 

p.47, The analytical measured exposure levels are so 

close to the target levels that it is inappropriate to 

use “reasonably” as a modifier. Please delete.

p. 48 GC Component identification: Isn‟t it 

necessary to describe the extraction efficiency 

correction factor of 23.94% used to accurately 

determine TBA content, as was specified in Appendix 

S and as was done in the BG+EtOH report? Please 

add appropriate information.

Revised to “acceptably close, in the 

opinion of the study director”.

Similar information as in G/ETOH report 

added to this report.

Accepted

p. 48

3.3.2 Weekly Clinical Observations (p.49): 

According to QA, data indicates yellow ano-genital 

staining was observed during recovery in a few high 

dose females on days 97, 104, 111. The text states 

that no staining was observed during recovery. Please 

check these observations and revise text as 

appropriate, especially since ano-genital staining was 

addressed by Dr. Schlesinger.

Revised that observations “abated” 

during recovery period.

Accepted

p. 50

3.5 Body Weights (p. 50, Fig. 2 & 3, Tables 4 & 5): 

This section needs to be expanded to reconcile the 

visual expression of decreased body weight in 

This section redrafted to indicate that 

“there were no adverse exposure-related 

differences....”

p. 51
211(b) Toxicology Research Group Gasoline TBA VC:

13- Week Subchronic Toxicity and FAB/MA Neurotoxicity 

Reviewer Checklist 

7 5/25/2012

Comments Action/Response Page

female rats [fig. 3 although never low enough to 

be statistically significant, with the statements in 

the text. Although the values may not be 

statistically or toxicologically significant, they can 

be considered indicators of exposure. Peer 

reviewers requested more discussion of weight 

changes.

Accepted

3.7.2 Functional Observational Battery (p. 51, 

Tables 7 & 9):

FOB Continuous: After reviewing the text in 

Table 7 which is reproduced in the report text, 

some rewording of the available information 

seems needed. 

 What is the significance, if any, that residuals 

from the observed foot splay were not normally 

distributed but the Blom transformed residuals 

were normally distributed? 

 Table II embedded in Table 7 apparently 

illustrates the conclusions that Blom 

transformed data for controls at wk 4 and 8 was 

significantly different from mid dose data. 

However, no significance indicators are given. 

The only asterisks in this table are located by 

control and low dose values which are cited as “ 

part of a two-way interaction, p <0.05”. Please 

explain.

 On p. 51 of report text, please indicate that 

“mean response of the control group was 

significantly different (higher) from the 

corresponding change in the mid dose group, but 

changes in the low and high dose groups were 

similar to controls.

The Statistical Report by Mark Nicolich 

reviewed and amended and the main 

report Summary and R&D updated as 

needed:

 If the observed data are skewed, as 

in foot splay data, the residuals from the 

analysis will not have a normal distribution. 

The Blom transformation of the data often 

results in residuals that are normally 

distributed. The main „significance‟ is that 

the observed data are skewed. 

 Rewritten based on new data and 

interpretation

 Rewritten based on new data and 

interpretation

Accepted

p. 52

p. 126

3.8.1 Hematology (p.52, Table 10): Some revisions 

to this section should be made since both peer 

reviewers considered changes in WBC parameters 

and differential counts to be attributable to 

BG+TBA exposure. I agree with the report that 

these changes are unlikely to be toxicologically 

significant but may be considered indicative of 

exposure and should be discussed in the text.

Upon further review by our Clinical 

Pathologist, we have decided that the 

original conclusion of „no effect‟ are still 

valid and therefore the text not revised.

Accepted

p. 53

3.8.2 Coagulation (p.52, Table 11): Text is not 

fully accurate about occurrence of significant 

increases in PT and APTT in all exposed males at 

week 4. In Table 11, only 2000 and 20000mg/m3

exposed males had statistically significant changes 

Section clarified that only the low and 

high exposure animals showed 

differences.

Accepted

p. 53
211(b) Toxicology Research Group Gasoline TBA VC:

13- Week Subchronic Toxicity and FAB/MA Neurotoxicity 

Reviewer Checklist 

8 5/25/2012

Comments Action/Response Page

in APTT at week 4; the 10000mg/m3values were 

similar to controls. The increase in PT was only 

statistically significant (p<0.05) in the 2000mg/m3

exposed males although the 20000mg/m3values 

were somewhat elevated. Please review these 

entries (also cited by QA).

3.8.3 Clinical Chemistry (p. 52): Dr. Schlesinger 

requested supporting information to the statement 

that “serum enzyme decreases are not generally 

considered indicative of test substance-induced 

toxicity”. Please provide references.

4.0 Conclusions: Add a sentence indicating that 

increased kidney weights (20000 and 

10000mg/m3) and histopathological changes in 

20000mg/m3 male rat kidneys are consistent with 

and indicative of hyaline droplet nephropathy. Dr. 

Goldsworthy requested these caveats because 

Mallory-Heidenhain stain was not employed to 

verify the presence of alpha 2-microglobulin.

This is based on tables in Gad et al, 

“Statistics and Experimental Design for 

Toxicologists”, 1988.

Added to conclusion in Summary and 

R&D.

Accepted

p. 54

p. 57

211(b) Research Group QA/QC Reviewer

1. Quality Assurance Statement, .p 4, - The 

protocol and report indicate that HLS was 

responsible for ensuring the GLP compliance 

status of several subcontractors (BioReliance, 

Immunotox, Inc. and Eye Research Centre). 

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. 

QA statements updated to reflect audits at 

subcontractors.

HLS did not 

do any of their 

own site visits 

or inspections

to assess the 

GLP 

compliance of 

the 

subcontractors, 

so there are 

none to report. 

They, as well 

as API must 

rely on the inhouse QA of 

these 

subcontractors 

to verify GLP 

Compliance. 

The QA 

Statement for 

the 

contractor‟s 

GFAP report 
211(b) Toxicology Research Group Gasoline TBA VC:

13- Week Subchronic Toxicity and FAB/MA Neurotoxicity 

Reviewer Checklist 

9 5/25/2012

Comments Action/Response Page

should be 

taken out as it 

is blank and 

will not be 

completed. 

2. GLP Compliance Statement: This page of the 

report was removed during the audit. 

Updated as needed and replaced into the 

report.

Ok

3. Summary, p. 5, second paragraph, next to last 

sentence: The text indicates that no 

histopathological evaluations were made at 

the Recovery interval. The data indicates that 

gross lesions for recovery animals were 

histologically processed and evaluated. This 

should be clarified in the report.

Added to the Summary. Ok

4. Summary, page 6, second paragraph, next to 

last sentence: Is there something missing 

from this sentence. It does not appear to make 

sense. 

Sentence deleted. Ok

5. 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 date of animal receipt for the genotoxicity 

animals (July 15, 2002) and dates of terminal 

sacrifice for immunotoxicity (July 23, 2002), 

genotoxicity (August 22, 2002) and GFAP 

(September 25, 2002) animals.

Not necessary since these other dates are 

detailed in the satellite reports and were 

not included in this section in prior 

reports in this series of 211b reports.

Ok

6. 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/

These minor differences were not 

considered protocol deviations thus the 

study data and report OK as is. The SD 

did sign in the data to accept the animals 

to go on test.

Ok

7. 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 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.

8. Methods, Section 2.5.7, Acclimation Period, These minor differences were not ok
211(b) Toxicology Research Group Gasoline TBA VC:

13- Week Subchronic Toxicity and FAB/MA Neurotoxicity 

Reviewer Checklist 

10 5/25/2012

Comments Action/Response Page

p. 18: The text states that animals were 

acclimated for approximately two weeks, 

which was the protocol-specified acclimation 

period. The actual length of acclimation was 

19 days for main study, neuropathology, 

GFAP and 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.

considered protocol deviations thus the 

study data and report OK as is. The SD 

did sign in the data to accept the animals 

to go on test.

9. Environmental Conditions: The Actual 

Temperature range was 22-26, as 21 degrees 

was only noted on a GFAP day and data from 

that day are not reported here. Some 

frequency and duration of the 26 degree 

readings would also be helpful.

GFAP information is included in this 

section. Elaboration of excursions has 

not been done for other finalized reports 

in this series and therefore was not done 

for this one.

Ok

10. 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.

Clarified that “Animals being tested were 

not exposed on days of neurological 

evaluations...”

Ok

11. 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 description 

of the methods presented in the text. 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.

Reviewed and revised. Ok

12. Methods, Section 2.13.1, Functional 

Observational Battery, p. 30 – The data 

indicates that the temperature range during 

FOB evaluations was 22 to 25 degrees C; a 

range of 22 to 24 degrees C is reported. Also, 

the data indicates that the illumination range 

Ranges corrected. ok
211(b) Toxicology Research Group Gasoline TBA VC:

13- Week Subchronic Toxicity and FAB/MA Neurotoxicity 

Reviewer Checklist 

11 5/25/2012

Comments Action/Response Page

during FOB evaluations was 48 to 65 

footcandles; a range of 51 to 65 footcandles is 

reported. Reported ranges should be corrected 

to reflect the data.

13. Methods, Section 2.14.1, Hematology, p. 33 –

In the footnote regarding manual differential 

counts, the word “an” should be deleted.

Deleted. Ok

14. Methods, Section 2.17.4, Tissues Preserved 

and Examined Histopathologically, Table 1, p. 

39: The Table should indicate that gross 

lesions were examined for Groups II and III 

(an “X” should be placed in the appropriate 

table column).

Added. Ok

15. 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. Ok

16. Methods, Section 2.18, Neuropathology 

Sacrifice, p. 41, last paragraph – The text 

states that tissues with macroscopic lesions 

were examined in all neuropathology animals. 

No gross lesions were observed. Should the 

text be modified accordingly?

Clarified that “Tissues with macroscopic 

lesions (there were none) were to be 

examined in all animals.” 

Ok

17. Methods, Section 2.23, Protocol Deviations, p. 

46 – The noise level readings taken before and 

after FOB evaluations on October 21, 2002 

were 50 and 51 decibels, respectively, which 

were outside of the protocol specified range of 

55 to 65 decibels. This should be included as 

a protocol deviation in the study report.

Protocol deviation added to data and to 

report.

Ok

18. Chamber Monitoring, first paragraph: 

Chamber distribution data reported indicates 

that the H-8 location was consistently low in 

the IIA chamber. Please comment. 

Added that “the only exception was that 

distribution location H-8 (left front port) 

on the Group IIA chamber was 

consistently about 20% below the target 

concentration.”

Ok

19. Results, Section 3.3.2, Weekly Clinical 

Observations, p. 49: The text states that 

yellow anogenital staining was not seen 

during the recovery period. The data indicates 

that this observation was present in a few high 

dose females on days 97, 104 and 111 

(incidences of 4, 1 and 1, respectively). The 

text should be revised accordingly.

Revised that observations “abated” 

during recovery period.

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

13- Week Subchronic Toxicity and FAB/MA Neurotoxicity 

Reviewer Checklist 

12 5/25/2012

Comments Action/Response Page

20. Results, Section 3.8.2, Coagulation, p. 52: 

The second sentence states that significant 

increases in PT and APTT were noted in all 

test substance-exposed male groups at week 4. 

Table 11 indicates significant differences for 

the low and high dose groups, but not in the 

mid-dose. The text should be revised 

accordingly.

Section clarified that only the low and 

high exposure animals showed 

differences.

Ok

21. Results, Section 3.10.2, Microscopic 

Pathology, p. 54: 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.) 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”.

Added. Ok

22. Results, Section 3.10.2, Microscopic 

Pathology, p. 54: The text (lines 4-5) 

indicates that no examinations were 

considered necessary for the recovery animals. 

The pathology report indicates 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 

otherwise documented in the study records. It 

is recommended that documentation 

authorizing the examination of the lesions be 

included in the study file.

Clarified R&D that no exams “(other 

than gross lesions)” were needed at 

Recovery. The protocol amendment#5 

has been drafted to clarify that gross 

lesions were to be read at Recovery.

Ok

23. Table 3, Ophthalmology Reports – The 

ophthalmologist‟s report for the 

neurotoxicology animals (dated September 18, 

2002) states that none of the ocular 

abnormalities were attributable to 

administration of the test compound. Since no 

ocular abnormalities were observed in these 

The Ophthalmology Report for the main 

study animals states that “none of the 

ocular abnormalities were attributable to 

administration of the test compound” 

while the Ophthalmology Report for the 

neurotox study animals states that “no

abnormality was seen”. Both reports 

ok
211(b) Toxicology Research Group Gasoline TBA VC:

13- Week Subchronic Toxicity and FAB/MA Neurotoxicity 

Reviewer Checklist 

13 5/25/2012

Comments Action/Response Page

animals, should the ophthalmology report be 

revised accordingly?

correctly correlate with the data.

24. Appendix A, the temperature and humidity for 

9/11/02 is noted with a footnote “a”. I could 

not find in this appendix where that is defined. 

The gauge fell and the battery fell out and was 

in the bottom of the chamber for the day, with 

no temperature/humidity recorded for that 

day. 

Footnote revised Ok

25. Appendices D and Table 4, Individual and 

Mean Body Weights – Week 17 body weights 

were recorded for all Recovery animals on 

October 22, 2002 (manually recorded data), 

but are not included in the report appendix and 

table. Should these body weights be reported?

Week 17 data has been added to mean 

and individual table/appendix for both 

absolute body weights and body weight 

change from baseline.

Ok

26. Table 7, Draft Statistical Analysis Report for 

FOB and Motor Activity – The following 

items were noted:

Mark Nicolich reviewed and:

a. Page 1, last paragraph, line 3 - it 

appears that the phrase “prior to” 

should be inserted before “start of the 

study”.

Done Ok

b. Page 2, Table 1 – Under Reflex 

Assessments, pinna reflex is not 

included in the list of evaluations 

performed. Sensitivity to light touch 

is included. Is this considered the 

same as pinna reflex?

Light touch changed to pinna reflex Ok

c. In the table entitled “Mean , sd and 

Sample Size for Beam Breaks per 

Interval by Sex , Study Week and 

Exposure Group”, the means and 

standard deviations reported for the 

recovery groups do not agree with 

those presented in Appendix G 

(Individual Motor Activity Data) or 

Table 8 (Mean Motor Activity 

Values). The data should be 

rechecked and tables and associated 

graphs and statistical analyses 

corrected as necessary.

The numbers (means, sd, n) agree among 

the 3 tables. The only difference I found 

was that means in the table entitled 

“Mean , sd and Sample Size for Beam 

Breaks per Interval by Sex , Study Week 

and Exposure Group”, were reported to 2 

decimal places while the means in the 

other tables were reported to 1 decimal. 

The means are reported to 2 decimal 

places to agree with the number of 

decimal places reported for the standard 

deviation.

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

13- Week Subchronic Toxicity and FAB/MA Neurotoxicity 

Reviewer Checklist 

14 5/25/2012

Comments Action/Response Page

27. Appendix G and Table 8, Individual and Mean 

Motor Activity Values: The means and 

standard deviations reported in the table and 

appendix for the Group IV females at Week 4 

appear to be incorrectly calculated. The 

calculated values should be rechecked and the 

table, appendix and any associated graphs 

corrected as needed. The statistical analysis 

report should also be checked to determine if 

statistics were performed on the correct mean 

values.

Table and appendix have been revised as 

needed. Statistical analysis report is not 

affected.

Ok

28. Appendix G and Table 8, Individual and 

Mean Motor Activity Values: Incorrect motor 

activity values are reported for Group IV 

recovery male 4055. The values reported for 

this animal appear to be a repeat of the results 

for the preceding animal. The report appendix 

should be revised to reflect the correct values 

for this animal and the corresponding 

summary table, graphs 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.

Table and appendix have been revised as 

needed. Statistical analysis report 

(including graphs) is not affected.

Ok

29. 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.

Appendix revised and is now consistent 

with summary table.

Ok

30. Appendix H and Table 9, Individual and 

Summary of FOB Evaluations – The 

categories reported in the table for Handling 

Evaluations, Ease of Handling differ from 

those presented in the Key for Appendix H 

and the key included in the study data. The 

table presents categories of: Normal; 

Moderately difficult; Freezes; Difficult; and 

Totally limp, corresponding to scores of 1 

through 5, respectively. The key indicates 

Table 9 revised and is consistent with 

appendix and data.

ok
211(b) Toxicology Research Group Gasoline TBA VC:

13- Week Subchronic Toxicity and FAB/MA Neurotoxicity 

Reviewer Checklist 

15 5/25/2012

Comments Action/Response Page

categories of: Very easy, totally limp; 

Normal; Moderately difficult; Freezes; and 

Difficult, corresponding to scores of 1 through 

5, respectively. The table and/or key should 

be revised to accurately and consistently 

categorize the responses for this parameter 

and should also be consistent with the data 

and SOP for this procedure.

31. Appendix H and Table 9, Individual and 

Summary of FOB Evaluations – Group III 

female 3566 was noted as having a right 

hindlimb injury at the time of the Week 8 

FOB evaluation. As per a footnote in the 

appendix, the air righting reflex result for this 

animal was excluded from group mean 

calculations. Would other parameters such as 

hindlimb grip strength and landing foot splay 

measurements also be affected? Should these 

values also be excluded from group 

calculations? The appendix, summary table 

and statistical analyses should be 

corrected/clarified as appropriate.

Only the data for air righting had this 

notation. Other data was collected and is 

reported.

Ok

32. Appendix H, Individual FOB Evaluations –

The following errors were noted during audit 

of the appendix against the raw data. The 

appendix will need to be corrected to reflect 

the raw data and the corresponding summary 

table (Table 9) revised accordingly. Statistical 

analyses may also need to be rerun. It is 

recommended that additional review of the 

appendix be conducted to ensure that all data 

are correctly reported.

See entries below:

a. Week 4, Group IV male 4075, Open 

Field Evaluations – The number of 

urine pools is reported as 2 in the 

appendix. The data indicates 0 for 

this parameter.

Table and appendix have been corrected. Ok

b. Week 4, Group II female 2568, Home 

Cage Evaluations – For palpebral 

closure, a score of 2 (eyelids 

completely closed) is reported in the 

appendix. The data indicates a score 

of 1 (eyelids open).

Table and appendix have been corrected. Ok

c. Week 13, Group IV female 4576, 

Open Field Evaluations – The number 

of urine pools is reported as 3 in the 

appendix. The data indicates 0 for 

this parameter.

Table and appendix have been corrected. Ok
211(b) Toxicology Research Group Gasoline TBA VC:

13- Week Subchronic Toxicity and FAB/MA Neurotoxicity 

Reviewer Checklist 

16 5/25/2012

Comments Action/Response Page

d. Week 13, Females – Incorrect body 

weights are reported in the appendix 

for Group I females 1576, 1577 and 

1578; Group II females 2566 and 

2567; Group III females 3566, 3567 

and 3568; and Group IV females 4566 

and 4567.

Table and appendix have been corrected. Ok

e. Week 13, Group III females 3566 and 

3567 – The forelimb and hindlimb 

grip strength and landing foot splay 

measurements reported in the 

appendix for these animals do not 

agree with the measurements recorded 

in the raw data.

Table and appendix have been corrected. Ok

f. Week 13, Females – “Tremors upon 

handling” were noted in the data for 

Group II female 2562 and Group III 

female 3563 in conjunction with air 

righting reflex and landing foot splay 

assessments. This observation is not 

reported in the appendix (it is 

footnoted for several other animals).

Footnotes added as needed. Ok

33. Table 9, Summary of FOB Evaluations, 

Pretest - For Group I Females, the reported 

incidences for Pinna Reflex do not agree with 

the data presented in Appendix H. The 

summary table reports incidences of 1 and 9 

for categories 1 and 2, respectively, whereas 

the appendix indicates incidences of 0 and 10, 

respectively. The statistical analyses may also 

be affected.

Individual information for Animal No. 

1577 has been corrected to “1”; summary 

and statistical analysis report are 

unaffected.

Ok

34. Table 9, Summary of FOB Evaluations, Week 

13 Males and Females – Many of the means 

and standard deviations for body weights, grip 

strengths and/or landing foot splay in the 

statistical analysis printouts do not agree with 

those reported in Table 9. The values reported 

in Table 9 appear to be correct. The statistics 

should be rechecked and re-run as needed.

Values checked and statistical analysis 

re-run. 

Ok

35. Table 9, Summary of FOB Evaluations, Week 

13 – The following incidences reported in the 

summary table do not agree with the data 

presented in Appendix H. The table should be 

corrected as appropriate. Statistical analyses 

may also be affected. It is recommended that 

additional review of the table be conducted to 

ensure that all data are correctly reported.

Table or appendix corrected as need; 

statistical analysis report will be checked.

a. Ease of Removal, Group II Males – Table corrected ok
211(b) Toxicology Research Group Gasoline TBA VC:

13- Week Subchronic Toxicity and FAB/MA Neurotoxicity 

Reviewer Checklist 

17 5/25/2012

Comments Action/Response Page

The summary table reports incidences 

of 10 and 0 for categories 1 and 2 

respectively. Appendix H indicates 

incidences of 8 and 2, respectively.

b. Ease of Handling, Group II Males –

The summary table reports incidences 

of 8 and 2 for categories 1 and 2, 

respectively. Appendix H indicates 

incidences of 0 and 10, respectively.

Table corrected Ok

c. Ease of Handling Group III and IV 

Males – The summary table reports 

incidences of 10 and 0 for categories 1 

and 2, respectively for each of these 

groups. Appendix H indicates 

incidences of 0 and 10, respectively.

Table corrected Ok

d. Locomotion, Group IV Females – The 

summary table reports incidences of 

10 and 0 for categories 1 and 2, 

respectively. Appendix H indicates 

incidences of 9 and 1, respectively.

Appendix corrected Ok

e. Arousal, Group IV Females – The 

summary table reports incidences of 

10 and 0 for categories 1 and 4, 

respectively. Appendix H indicates 

incidences of 9 and 1, respectively.

Appendix corrected Ok

f. Pinna Reflex, Group II Females – The 

summary table reports incidences of 2 

and 8 for categories 1 and 2, 

respectively. Appendix H indicates 

incidences of 1 and 9, respectively.

Table corrected Ok

36. Appendix I, Individual Hematology Values: 

Manual differential counts were performed 

and reported for a few animals, including: 

Group III female 3551, Week 4; Group II 

female 2557, Termination, and Group I female 

1555, 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 indicate that 

“absolute large unstained cells” are not 

evaluated in manual differential counts and 

are reported as 0 in these instances. This 

notation appears on the Preface page for 

Appendix K (Individual Clinical Chemistry 

Values), but should be moved to the 

Hematology preface page.

Appendix I preface to add appropriate 

note, and note removed from Appendix 

K.

ALUC note 

still appears on 

Appendix K 

Preface. 

Appendix I 

was corrected .

37. Appendix K, Individual Clinical Chemistry Note removed as unnecessary. Highlighted 
211(b) Toxicology Research Group Gasoline TBA VC:

13- Week Subchronic Toxicity and FAB/MA Neurotoxicity 

Reviewer Checklist 

18 5/25/2012

Comments Action/Response Page

Values, Preface – A notation at the bottom of 

the page indicates that LD for animal 2553 at 

Week 4 was QNS to repeat spurious results 

and the original value was reported. This 

same situation occurred in some other 

instances including animal 1562, Week 4 ALT 

and AST and animal 1562, Termination, Na+. 

Should the notation be revised to include all 

similar instances? Also, for animals 3559 and 

4561, Week 4, a notation on the preface page 

indicates that due to low sample volumes, bile 

acid analyses were not repeated using diluted 

samples. It is recommended that this notation 

be revised to explain that the results for these 

animals were reported as >200 umol/L (above 

the limit of quantification).

sentence not 

removed. 

Note on 

Preface page 

indicates 

>200=above 

the limit of 

quantitation 

(could not be 

diluted into 

range). It 

should appear 

with Bile 

Acids footnote 

and have units 

of measure 

included.

38. Appendix M, Individual Gross and 

Microscopic Observations and Table 14 

Summary of Gross Necropsy Observations –

For Group 4 female 4562, Appendix M 

reports a gross observation of enlarged 

mediastinal lymph node, which was noted 

post fixation. The Histolotechnology Record 

sheet documents that this observation was for 

the mesenteric (not mediastinal) lymph node. 

The appendix and summary table should be 

corrected to reflect the raw data.

Pathology report has been corrected to 

reflect the data.

ok

39. Appendix M, Individual Gross and 

Microscopic Observations – For 

neuropathology animal 4071, a notation on the 

individual microscopic pathology report 

indicates that the sural nerve was missing 

from the slide and was recut. The 

Histotechnology Record for this animal 

indicates that the sural nerve was missing at 

the time of initial processing and therefore, no 

recut could be made. It is recommended that 

the pathology report be clarified to indicate 

that this tissue was missing.

Pathology report has been clarified to 

reflect the data.

ok

40. Appendix S, Analytical Report: It should 

probably be made very clear that the 

correction factor is only used in the one 

column of data reported and highlighted on 

page 8. All other chamber data reported has 

not been corrected. 

It has been clarified. ok

41. Appendix 4, Analytical Report: Page 9: The 

data for Sample 1001B is labeled 1001A also. 

Corrected to 1001B. ok
211(b) Toxicology Research Group Gasoline TBA VC:

13- Week Subchronic Toxicity and FAB/MA Neurotoxicity 

Reviewer Checklist 

19 5/25/2012

Comments Action/Response Page

Please verify that 1001B is correct and correct 

data. 

42. Appendix 4, Analytical Report: Page 15: 

Some samples were run on 8/12, after there

was a sample transfer interruption. It is 

indicated that this interruption did not affect 

the integrity of these runs since 4 day‟s inprocess stability has been established. It 

appears that there was only one other time 

when 4 days between receipt and analysis 

(during pretest trials) occurred and this 

instance is actually five days between receipt 

and analysis. Please comment. 

The in-process stability actually referred 

to the time period of the samples being 

kept on the autosampler. The first set of 

injections including the spiked charcoaltubes, test material standards and some 

samples were made on 8/8/02 and the 

injections were resumed on 8/12/02, 

which was within the 4-day in-process 

stability period.

ok

43. Appendix T, Neurobehavioral Positive 

Control Data - The neurobehavioral positive 

control evaluations were performed as 

separate in-house studies at HLS under Study 

Nos. 00-14500 (FOB Study) and 00-14501 

(Motor Activity Study). The reports included 

in Appendix T of Study No. 00-6131 are 

abbreviated versions of the final reports of the 

separate in-house studies. Appendix T 

includes no reference to these individual study 

numbers. This information should be 

included to clarify that the positive control 

evaluations were not conducted as part of API 

Study No. 00-6131.

Appendix T has been replaced with updated neurobehavioral positive control 

data.

Ok 

Additional Raw Data Comments

1. Feed Filler Preparation Records – A memo to 

the file indicates that documentation of feeder 

filling is missing for the weeks of June 18 and 

25, 2002 and July 2, 2002. The memo states 

that these records will be placed in the study 

file when found. However, it does not appear 

that the missing records were found. If the 

records have not been located, this should be 

addressed as a deviation in the study records 

and final report.

The data has not been found but the 

records do show that the animals were 

properly fed during these intervals thus 

not a protocol deviation. However, an 

SOP deviation has been added to the 

data.

ok

2. Blood Collection Records – The method of 

blood collection (orbital sinus puncture) does 

not appear to be documented anywhere in the 

data. It is recommended that this information 

be added to the study records.

We could not find any record as to blood 

collection site. We added this note to the 

animal fasting and transfer record: 

“Method of blood collection was not 

documented at any interval. It appears as 

if orbital sinus was used”. It was assumed 

we performed procedure as per protocol.

ok

3. In-Chamber Observations for Chamber IVB – Corrected to 22July02. ok
211(b) Toxicology Research Group Gasoline TBA VC:

13- Week Subchronic Toxicity and FAB/MA Neurotoxicity 

Reviewer Checklist 

20 5/25/2012

Comments Action/Response Page

For Exposure Day 20, the date, which is 

recorded as a late entry, is written as July 23, 

2002. The date for Exposure Day 20 was 

actually July 22, 2002.

4. Animal Assignment/Randomization Records, 

Genotoxicity Groups – The printout of 

animals eliminated from assignment 

documents that temporary animal 14 was used 

for training purposes on September 25, 2002, 

but does not clearly indicate the disposition of 

the other animals not assigned to study (i.e,

temporary animals 31, 24, 52, 40 and 35). 

This should be addressed in the data.

The final disposition was not documented 

for these extras. We added this comment 

to the form.

ok

5. Serum Chemistry Data, Week 4 – For animals 

1566 and 2556 (sample ID numbers 46471 

and 46481, respectively), notation “5” is 

recorded next to the sample identification 

number on the data monitor report. The 

corresponding footnote on the Hitachi 717 

worksheet indicates “See page 15 for dilution 

due to low sample volume” and Page 15 of the 

data monitor report shows a diluted sample 

run for LD only. It is not clear from the 

notations on the original data monitor report 

and Hitachi Worksheet that only this one 

parameter was to be diluted and re-run. It is 

recommended that this be clarified in the data.

Clarification was added to the Hitachi 

717 worksheet page to address this issue.

ok

6. Serum Chemistry Data, Week 4 – The original 

data monitor report for sample ID no. 46491 

(animal 3556) should have an appropriate 

footnote to indicate that phosphorus was rerun to confirm the high value.

A footnote was added to the Hitachi 717 

worksheet page to address the repeat 

phosphorus.

ok

7. Serum Chemistry Data, Recovery – For 

sample ID nos. 50688 and 50689 (animals 

1051 and 1052), CREAT, GGT, GLU and 

AST results all have footnote “1” recorded on 

the data monitor report with the corresponding 

notation “NV, See page 12 for result due to 

instrument flag” written on the Hitachi 717 

worksheet. However, no instrument flag 

appears on the original data monitor report for 

these parameters. This should be clarified as 

appropriate in the study records.

Another footnote was added to the 

Hitachi 717 worksheet to explain the 

repeating of these analytes.

ok

8. Serum Chemistry Data, Recovery – For 

sample ID no. 50694 (animal 1058), Footnote 

“11” on the Hitachi 717 Worksheet indicates 

that due to equipment malfunction, tests were 

performed in order of priority per the protocol 

A reagent probe movement error 

occurred during the analysis. As a result 

any analyte tested for this sample was 

lost. This issue was remediated and 

documented separately on a non routine 

ok
211(b) Toxicology Research Group Gasoline TBA VC:

13- Week Subchronic Toxicity and FAB/MA Neurotoxicity 

Reviewer Checklist 

21 5/25/2012

Comments Action/Response Page

and remaining tests reported as QNS. The 

notation does not indicate the nature of the 

equipment malfunction. It is recommended 

that this be clarified in the data.

maintenance log in the instrument 

maintenance log book. A foot note was 

added to the worksheet to refer to the non 

routine maintenance log.

9. Summary of In-Life Data (Confirmation of 

Last Physical Observations) –The forms 

inconsistently document confirmation or nonconfirmation of the observation “Nasal 

discharge, red”. In most instances, 

confirmation of this finding at necropsy is not 

documented on the forms (see animals 3056, 

3059, 4065, 4066 and 4562 for example). In 

other instances, (see animals 1561, 1563 and 

3553), and “N” indicating that the observation 

was not evident at necropsy is recorded on the 

form. These inconsistencies should be 

addressed/clarified in the study data.

These have been addressed in the data 

and SOP deviation memo signed and 

added to the study file.

ok

10. Necropsy Slide Fixation Record – For 

recovery sacrifice animal 4051, the number of 

slides prepared is not documented on the 

form. An entry of “1‟ should be recorded.

This has been addressed in the data and 

SOP deviation memo signed and added to 

the study file.

ok

11. Histotechnology Record Sheet, Animal 3053 –

The histology record does not indicate that a 

gross lesion was present in the lung of this 

animal. It appears that the notation “LR” 

(lesion taken in routine section) should have 

been recorded at the time of tissue trimming. 

The post-fixation observation has been 

entered and the entry verified.

ok

12. The large cylinder usage for tank 1B2 on 5/29 

is 8102 g., but it is unclear where this value 

came from.

Based on review of the data, this value 

corrected to 8100 grams.

ok

13. GFAP assay data: The ug protein loaded for 

Hippocampus – the original print-out is lower 

for mean values than subsequent print-outs. Is 

the difference in rounding? Animals 

#2,4,5,7,8, and 11, are examples. 

Yes – rounding is the difference. ok

14. GFAP assay data: The Rest of Brain, GFAP 

assay, worksheet, the date of assay is needed. 

Same is true of the Hippocampus, GFAP 

assay. 

Dates have been added. ok