### 211(b) Toxicology Research Group Gasoline Tame VC: A 13- Week Whole Body Toxicity Study in Rats with Neurotoxicity
Assessments and 4-Week In Vivo Genotoxicity and ImmunotoxicityAssessments
Reviewer Checklist

1 10/16/2009 

### Inhalation Toxicity Report Comments Action/Response Page 211(b) Research Group QA/QC Reviewer

The following item re quires consideration:

2. Compliance Statement : Since it is the sponsor’s responsibility to maintain the method of synthesis, fabrication, or derivation of the test fuel, and this has not been completed, it must be included in the sponsor’s compliance statement. Added to Compliance Statement. Needs to be updated with latest sentence used in other reports. (information was not available during study, but is now maintained with sponsor) Compliance statement up-dated as indicated.

4. Page 4, Quality Assurance Statement: Since HLS has inspected all of the subcontr actors (except CDC) these facility inspections should be listed on the QA statement for this study (Immumotox and BioReliance periodic inspections). Our inspections of the Subcontractors added to our included QA Statements. I don’t see inspections of these two facilities. Those
listed are for work done at HLS. Facility inspections are listed on the HLS QA
statements contained within each sub-contract
report.

6. Page 7, last sentences , first paragraph: There was a variation noted during Exposure 13. It is noted as “suspicious” on the table, but no mention of it occurs anywhere. Unless it is known to be in error, the variation should be reported. We don’t find any ‘suspect’ values noted in the CMR table in Appendix A for Exposure 13 or any other exposure day. Sorry, this was in the analytical report. Appendix S... Exposure 13, footnote indicates suspect
values. Not noted whether included in mean or not. Was it, and should it have been? The sample is included in the mean in the Summary Table. The word “suspect” is removed from the report (Exposure 13 table)
to avoid confusion.

11. Page 47, Chamber monitoring table : Group 4,
nominal concentration standard deviation is 661 in other
tables. Corrected. I cannot verify. Starting on page 49 of the text of the report (Results and Discussion), pages from ETOH are included. This continues until page 62, so I can’t verify changes through item #17. In scanning through the appendices, it has come to our attention that not only the text of the report, but several of the Appendices have Ethanol
Tables. The TAME Micronucleus and the Sister Chromatid Exchange reports contain tables from the Ethanol report. At least the headings of the tables indicate ethanol. We assume/hope that this is just an error in collecting all of the sections of data for the reports, but request that HLS QA check to make sure all appropriate tables, appendices and text appear in the next version of this

### 211(b) Toxicology Research Group Gasoline Tame VC: A 13- Week Whole Body Toxicity Study in Rats with Neurotoxicity
Assessments and 4-Week In Vivo Genotoxicity and ImmunotoxicityAssessments
Reviewer Checklist

2 10/16/2009 

### Inhalation Toxicity Report Comments Action/Response Page

report. Report originals checked and correct R/D is present; incorrect (00-6127) R/D does however appear to be in the PDF file

submitted for 2 nd draft. In-life tables presented with micronucleus and SCE reports were the correct files, though it appears that an error was made in the entry of the TASC protocols in as much as the wrong test substance was listed in the title. The study title was subsequently corrected and the tables have been reprinted. The remainder of the report was checked and all pages presented are for the indicated study (TAME).

12. Page 48, last paragraph : data were consistent from week-to-week except for exposure 13, where ther e should be some mention of the “suspicious” values for the group three
chamber. We don’t find any ‘suspect’ values noted in the CMR table in Appendix A for Exposure 13 or any other exposure day.

13. Page 50, Motor Activity : The statement that the subsequent statistical evaluation of the data did not confirm this initial determination, made in the last sentence is not altogether true. There were some statistically significant findings in the 13th week data. They should be discussed
here. The Mark Nicolich text has been added here although the conclusion of no effect has not changed.

14. The draft report, on page 51, section 3.8.3 Clinical Chemistry discusses the parameters that were statistically significant. The dosage levels should be added to this section to clarify these results. (alkaline phosphatase, males 20000 mg/m 3 ; chloride, females 10000 and 20000mg/m 3 ) Exposure levels have been added.

15. The draft report, on page 51, section 3.8.3 Clinical Chemistry discusses the terminal sacrifice results. In the third sentence of this section, male animals in the 20,000mg/m 3 level should be added and the fourth sentence should add in the 10,000 and 20,000 mg/m 3 levels to clarify
the results. Exposure levels have been added.

16. The draft report text on page 52, section 3.9 Organ Weights and Brain Measurements should be reviewed to clarify the discussion on liver weights. Only three liver weights were statistically significant: Male relative to body weight, 20,000mg/m 3 , terminal sacrifice; female, absolute weight, 20,000mg/m 3 recovery sacrifice; male relative to body weight 20,000mg/m 3 recovery sacrifice. The current text is adequately descriptive and the reader may review the tables for more
details for these no effects differences.

17. Page 52, Macroscopic Pathology : It may be helpful to indicate that 6 of 20, meanin g 3 main study animals and 3 recovery animals, exposed to 20,000.... Added to this text.

### 211(b) Toxicology Research Group Gasoline Tame VC: A 13- Week Whole Body Toxicity Study in Rats with Neurotoxicity
Assessments and 4-Week In Vivo Genotoxicity and ImmunotoxicityAssessments
Reviewer Checklist

3 10/16/2009 

### Inhalation Toxicity Report Comments Action/Response Page

20. Table 12 and Appendix L do not contain the Sponsor Study Number in the heading. The individual pages in Appendix L and Table 12 are not labeled as such. Headings will remain as is. The computer program does not allow for addition of a sponsor study number. Table 12.... 01-6128 on page 1 ... should be 00-6128 Typographical error in the heading of the preface page has been corrected.

### 211(b) Toxicology Research Group Gasoline Tame VC: A 13- Week Whole Body Toxicity Study in Rats with Neurotoxicity
Assessments and 4-Week In Vivo Genotoxicity and ImmunotoxicityAssessments
Reviewer Checklist

4 10/16/2009



### GFAP SATELLITE REPORT

Comments Action/Response Page

GFAP LEVELS IN SPECIFIC RAT BRAIN AREAS FOLLOWING A 13-WK WHO LE BODY INHALATION
EXPOSURE TO BASELINE GASOLINE VAPOR 

### Peer Reviewer – Schlesinger Peer Reviewer - Goldsworthy
Pituitary GFAP levels could only be detected in less than 25% of samples; due to the paucity of data and inability to make any conclusions from this region of the brain, this data should be omitted from Tables and noted in text that analysis of pituitary could not be adequately performed. WG agrees with the reviewer’s comments. Sparse pituitary data has been deleted from Tables 5 and 6, but retention of individual values in Table 7, pr imarily because this
series of studies is one of the first in which GFAP levels in pituitary have been measured. Although other 211(b) reports in the series show somewhat better recovery of pituitary data, this study has no usable data for females in control, low or high doses and only 1 entry at mid-dose; data for males is only slightly
better. To include this limited data in summary tables (5&6) gives an erroneous impression that sufficient animals/group responded. Keeping individual entries (Table 7) provides an idea of the level of GFAP likely to be present in this small organ, providing a starting point for improvement of assay methods. Results and conclusions can
also be revised to indicate that GFAP was not
increased in 8 areas of the brain, pituitary
excluded.

Done.

Note that text, page 18, states that ‘half of pituitaries’ gave results below level of sensitivity. This is incorrect according to Table 7, where the majority (9/40) of animals were shown
to be below limits of detection. Since the pituitary entry in Table 7 is retained, the text on p. 18 has been corrected

The footnote content on Table 7 should appear on Page 17 or 18 to clarify.

Done