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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, howeve r, 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 ov er to the rest of the report. Also, the 13 week time point is referred to as week 12 in the same

table. T he Stat istical 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. 1 23

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 l ow dose group decreased markedly from 4 to 8 weeks, which apparently was the key factor giving rise to the statistically significant interaction. Never

- the

less, the decreased foot splay observation was not dose related and peripheral neuropathies typical ly 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. T he 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 it s own

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

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Reviewer Checklist

2 5/25/2012 Comments Action/Response Page

level for a ll 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

cons istency 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 i n 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 wei ght 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 th at 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:

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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 (te mperature, 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 do es 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. Ani mals 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 cons umption 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:

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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 hig h 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 decr eased 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 wer e 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, Conclusion s, 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 al pha 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/m 3 exposure 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

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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

con sidered 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/m 3 and <5.0% at 20000mg/m 3 , and both values, especially analytical, were close to th e 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 e xposure

- 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 b e 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

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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 th e 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 Mallory

Heidenhain 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. De leted 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, espec ially 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

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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

statistic ally 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

transf ormed 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 intera ction, 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

s imilar 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 discus sed 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 accura te about occurrence of significant

increases in PT and APTT in all exposed males at week 4. In Table 11, only 2000 and 20000mg/m 3
exposed males had statistically significant changes Section clarified that only the low and high exposure animals showed

differences. Accepted p. 53

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Reviewer Checklist

8 5/25/2012 Comments Action/Response Page in APTT at week 4; the 10000mg/m 3 values were

similar to controls. The in crease in PT was only statistically significant (p<0.05) in the 2000mg/m 3
exposed males although the 20000mg/m 3 values

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/m 3 ) and histopathological changes in 20000mg/m 3 male rat kidneys are consistent with and indicative of hyaline droplet nephropathy. Dr.

Goldsw orthy 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 insp ections 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 in

house QA of

these

subcontractors to verify GLP Compliance.

The QA

Statement for

the

contractor‟s GFAP report

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Reviewer Checklist

9 5/25/2012 Comments Action/Response Page

should be

taken out a s 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 th at 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

p artial 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 genoto xicity 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

no t 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 slight ly 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

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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. T herefore, 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 o ne. 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 regul ar 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:

S ome 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

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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 co rrected

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 Hist opathologically, 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 Histopathologic ally, 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

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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” fro m 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 w ere 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 deci sion 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 fi le. 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 fo r 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

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Reviewer Checklist

13 5/25/2012 Comments Action/Response Page animals, should the ophthalmology report b e 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 chang e 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 (Indi vidual 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 re ported to 2 decimal places to agree with the number of decimal places reported for the standard

deviation. Ok

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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

statis tics 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 repo rted 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 tab le, 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

need ed. 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

shou ld 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; Fre ezes; 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

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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. Append ix 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

appen dix 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 appen dix 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 co rrected. 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

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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 an d

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 oth er 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

fo r 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 r eported 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

e nsure 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:

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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, respectivel y. 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 i ncidences 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; Gro up 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 indicat e 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

H ematology 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

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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, Terminat ion, 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 i s 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) l ymph 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 t he 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

r ecut 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

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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 in

process 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 charcoal

tubes, 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

Cont rol 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

abbreviat ed 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 up

dated 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

addres sed 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:

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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. T he 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 1 5 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 cl arified 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 re

run 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 mo nitor 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. 506 94 (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 rou tine ok

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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 equi pment 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 non

confirmation 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 ani mals 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 sig ned 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 deviatio n 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 sectio n) 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, a re 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