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EPA

Scientists have reviewed the submission dated November 20, 2006 from the Section 211(b) Research Group of the American Petroleum Institute regarding the reports reviewers’ comments and the Research Group responses for the 211(b) Draft Baseline Gasoline Vapor Condensate Chronic Carcinogenicity Study

and Draft Baseline Gasoline + MTBE Vapor Condensate Chronic Carcinogenicity Study. No response required. NA

In general, the API extramural reviewers found that the study was appropriate

conducted based on the current

recommendations for a chronic carcinogenicity study. In addition, the reviewers identified several important points with regard to presentation and additional small data needs. In response to those reviews the Research Group has identified the appropriate actions. No response required. NA

In addition, the NHEERL reviewers suggest that the following issues be considered by API in preparing a revised final report. See responses below. NA

Comments on Study FY01-013 GMVC

Page 3-4 [Abstract]. While there was not a great number of early deat hs in this study, the summary tables use 50 as the denominator.

This would underestimate the significance of a finding at the end of a study for low incidence events that occur late (say 600 days on study). For example renal tumors tend to be a late

event and it would be more in line with the

NTP if one were to use time to earliest tumor

as the cutoff for the denominator for the exposure groups in table 3-12 and 3-14. A denominator of all 50 animals is considered appropriate since there was no treatment-related

effect on survival and statistical analysis to

account for time of death (logistic regression)

was conducted.

Additionally, the study was conducted under Section 211(b) Alternative Tier 2 requirements

and EPA Health Effects Test Guidelines OPPTS 8700-4200 “Carcinogenicity.” Testing guideline Section (e)(1)(i) states:

(e) Data Reporting --(1) Treatment of Results. (i) Data should be summarized in tabular form, No change

1 Page numbers refer to pages in body of repor t and pathologist report in documents with suggested revisions. Once LRRI revises t he report, these number will need to corrected to the final page numbers. Also note, pa ge designation of text revision in response to comments was specifically requested by EPA.

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showing for each test group the number of animals at the start of the test, the number of animals showing lesions, the types of lesions, and the percentage of animals displaying each type of lesion.

Page 3-21, Table 3-16. Using mean severity score is the only way to compare as nephropathy would usually be a finding in 100% of rats in any chronic study (table O-3). Average severity scores have been added to table 3-17 (formerly Table 3-16). 3-29

Page 0-11. Top of page – a2u-globulin could

account for these results. Alpha-2u-globulin overload as a potential mechanism for induction of male kidney adenomas has been specifi ed in this discussion, since this mechanism has previously been associated with light hydrocarbons and MTBE in induction of renal adenomas, renal carcinomas and increased severity of nephropathy in male rats. P-14- P15

Page O-14. Last sentence of full paragraph in middle of page beginning ...However, - this is an incorrect interpretation. It is not possible to specifically differentiate chronic nephropathy from a2u from chronic nephropathy due to spontaneous CPN in a 2- year old male rat. If the lesions of linear mineralization are present in the animal they are typically associated with a2u but absence of that change does not rule out a2u contributing to the nephropathy. The protein a2u-globulin is no longer produced by the rat liver as the rat ages, usually after 1 year of age and so would not be expected to be present in the kidney of 2-year old rats. Therefore, it is not unexpected [ RG note: ‘unexpected’ should probably be ‘expected’] to find to hyaline droplets in an aged male rat with a2u-induced tumors. Nephropathy in the female rats, does not indicate anything about the contribution of

a2u in the males. Text in both the pathology report (Appendix P) and the body of the report has been revised to indicate that (1) CPN ca n mask a2u-globulin overload induced male nephropathy in older male rats; (2) a2u-globulin overload in the first year of life can increase the severity (not incidence) of male CPN; (3) a2u-globulin production by the male rat liver typically stops at ~ one year of age; (4 ) a2u associated hyaline droplets would not be expected after production of a2u-globulin ceases ; (5) a2u-globulin nephropathy is male rat specific and would not be involved in female CPN; and (6) due to the sex differences, female CPN effects cannot be used to substantiate any conclusions in males regarding the role of a2u overload in male CPN

and renal tumor induction. P-14 to P-15; 3-

29

The following references should aid in interpretation of these data:

• Licata et al. Tox Sci 62:191-204,

2001

• Williams and Borghoff. Tox Sci The RG and investigators thank EPA for providing these references to aid in data interpretation.

[ Note - Refs focus on MTBE as an a2u-globulin Cruzan reference

used. P-16.

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57:146-155, 2000

• Williams et al. Tox Sci 54:168-176,

2000

• Prescott-Mathews et al. TAAP 157:60-67, 1999

• Poet and Borghoff, TAAP 145:340- 348, 1997

• Prescott-Mathews et al. TAAP

143:301-314. 1997

• Borghoff et al. Fundam App Tox 30:264-275, 1996 inducer; MTBE as an inducer of testosterone biotranformation, altered endocrine responses & Leydig cell tumors; and MTBE & TBA PBPK

models in rats and MTBE PBPK model humans.

A recent review of MTBE tumorigenicity modes of action may also be of interest to the investigators; Cruzan et al., Reg. Tox. Pharm.

47 :156-165, 2007]

Peer Reviewer – Schlesinger

Overall, this was a clearly written concise report, except for the Conclusions. As an aside the variability of the three exposure atmospheres was almost identical, which is excellent for a study of this duration (Table 3- 2). There were no significant deviations from the accepted study protocol that would have affected the outcome. However, the conclusions and summary need to be modified so that the reader has a clear “picture” of changes induced by exposure and of the biological significance of these in relation to the goals of the study. As written, things

seem to be somewhat “muddled” and this reviewer, after, reading the report, could not decide whether the exposures did or did not result in biologically significant effects of potential concern to human health. The conclusions and summary have been modified to present a clearer picture of changes induced by exposure and of their biological significance. iii, S-3, 3-22

p. 2.3 Diet and Drinking Water. How did the investigators conclude that any contaminants

in the diet or water would not interfere with the study results? Were there any contaminants present and, if so, what were they? The methods section has been revised to indicate that the diet was analy zed by the manufacturers

for various contaminants and that established

maximum allowable concentrations were not

exceeded. It will also indicate that water analyses for heavy metals, pesticides, PCBs, nitrates and microbes are conducted routinely by an independent laboratory and that all analytes are sufficiently low as to not interfere with the outcome of the study. 2-4

p. 2-11. Statistics. Body and Organ Weights. It is noted that if data were homogeneous, that significance was assessed using a modified The methods section has been revised to indicate that ANOVA is run as part of the statistics package. The Path To x statistical package 2-12

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Dunnett’s test. This is a post hoc test to assess differences between groups after a primary statistical test, such as ANOVA, is performed. What primary test was used in this regard by the investigators? Furthermore, how was the Dunnett and t-test modified and, for the latter, was any correction used for multiple testing? discussion of test modifications was limited; information available will be provided.

p. 3-2. Confirmation of Absence of Aerosols. I would change the section title to Aerosol Concentration Analysis. In any case, were the particle measurements taken throughout theactual the exposure period or only in the preexposure calibration period? The Workgroup prefers the existing section title since the title “Aerosol Concentration Analysis” implies that aerosols are expected and are being

characterized. These measurements were taken during the chamber trials. No particle measurements were taken during the actual exposures since animal dander could contribute to particles in the exposure atmosphere.

Details on the TSI instrument use for particle analysis are now provided in Appendix E, page E-54. Actual data are provide on pages E-20 to

E-31. No limit of detection has been located. No change to pg 3-2. Appendix E, pages E-20 through E31 and pg E-54

p. 3-3. Gas Chromatographic Profiles of GMVC Exposure Atmospheres. It is noted that profiles remained “acceptably” constant through-out the study. What was the standard of acceptability? There was no rigorous standard of acceptability with respect to individual hydrocarbons. The Research Group is satisfied that the profiles generated and reviewed regularly demonstrated reproducible exposure atmospheres. No change

p. 3.3. Mortality. The difference among the groups for the females is very close to the accepted level of significance at p=0.07. Should the results thus be carefully assessed for actual biological significance or was the exposure data statistical analysis made artificially close to statistical significance

because the control values seem to be low

after 600d. Some further discussion is needed

about results that are close to statistical significance. In comparing the mean survival of control females in this study (666±11.5) with BGVC female control survival (687±11.4) and a previously conducted chronic inhalation study at this facility (682±10), GMVC control female survival was low. This would explain the low p value. The RG feels there is no biological significance for the higher mean survivals of the treated groups. No change

p. 3-9. Clinical Signs of Toxicity. Some summary table would be useful here. Since there were no treatment-related effects on clinical observations, a summary table is not

necessary. No change

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p. 3-12. Table 3-9. The value for lungs for Male Exposure Group 2 shows a mean larger than in groups 3 and 4, yet is apparently not statistically significantly different from control due to the larger SD. Some explanation is needed, since this appears to indicate a real change in organ to body weight at all levels of exposure. There were no significant increases in absolute lung weights in any treatment group (Table 8). The significant increases in the lung-to-body weights in Table 9 are consistent with the treatment-related decreases in body weight for the mid- and high treatment groups, and are considered to be caused by the treatment effects on body weight rather than treatment-related effects on lung weight. The text has been

revised. Table 8, pg 3-11; Table 9, pg 3-13 and text on pg 3-

12

p. 3-10. Percent Organ to Body Weight at

Final Sacrifice. It is noted that the “effects were exposure concentration dependent.” However, this should be stated as an “eyeball” observation, since there was not trend analysis or no testing of ex posure groups vs. each other, as opposed to exposure vs. control. No statistical test was performed to evaluate concentration dependence. The text has been

modified state that there is an ‘. . . apparent trend’ for exposure concentration dependence. 3-12

p. 3-15. Table 3-11. Was cell analysis performed at the lower exposure levels. Cell analysis was not performed at the low- and mid-exposure levels. No change

p. 3-14 Proliferative Lesions. Data for

testicular cell adenomas noted here does not

seem to be included in Table 3-12. The data for testicular cell adenomas is included

in Table 3-12. 3-18

p. 3-16. Table 3-12. The investigators should address the finding that the value for mononuclear leukemia for spleen at the high exposure level is not significantly different

from control while the same mean values at the other exposure levels are different from

control.

Is it a function of the differences in sample size at the low and mid concentration and, if so, what conclusions of biological significance should be made for this endpoint? The high incidence of le ukemia in low- and middose groups is likely a function of the low “n” since only spleens that appeared abnormal at gross observations were subjected to further analysis. It is likely the incidence in the mid and low dose groups would decrease if all spleens were analyzed. The Research Group appreciates the reviewer’s point about statistical significance and biological significance; however

mononuclear cell leukemia is a common and expected phenomenon in aging rats. No change

p. 3-16. Proliferative Lesions. It is noted that diagnoses were made on animals where gross lesions in the spleen were present and therefore there was some sampling bias. If so,

how would this have affected the conclusions and why was the study conducted in a manner This study was conducted in compliance with EPA OPPTS 870.4200 as specified in the Clean Air Act Section 211(b) regulation. Toxicology studies very commonly use protocols in which all tissues at the highest level of exposure are examined for abnormalities, and at the lower No change

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that included such bias? levels of exposure, only target tissues and tissues with gross findings are examined further. While this might introduce what the report and reviewer refers to as “sampling bias” and spurious incidences of st atistical significance at the lower exposure levels, the Research Group believes the high incidence of mononuclear leukemia in spleen is more likely related to the low “n” based on evaluation of only spleens that appeared abnormal at necropsy at the terminal sacrifice. Had a signif icant number of findings been present in the high exposure group, all tissues in the lower dose groups would have been examined, as instructed in the protocol. This explanation for protocol-driven bias in the mid-dose groups is included in the report text.

p. 3-18. Conclusions. In the third paragraph,

it is noted that “...effects seen at 12 months...were more likely due to difficulties in blood sampling than to GMVC inhalation.”

What is the scientific basis for this

conclusion? The difficulties in blood sampling at 12 months are explained on pg 3-15. No change

p. 3-21. Conclusions. A better summary paragraph is needed at the end of this section. The term “few differentiators” is too vague

and nonconclusive. This section of the conclusions has been revised to better describe the comparisons between the

health effects BGVC and GMVC. 3-22 thru 3-30

p. O-8. Appendix O. Pathologist Report. As noted above, why was there a “protocol driven sampling bias” and how would such a bias affect conclusions of health significance of the

test material? The explanation of protocol driven sampling bias is presented on pg P-11 and P-12. The biological significance of low- and mid-level sampling bias is that statistical increases in lesions found only at these two doses and not the high dose are spurious and not treatment related. No change, P-11,

P-12.

Peer Reviewer – Goldsworthy

I have reviewed the API sponsored draft final report “211(b) Chronic Carcinogenicity Gasoline MTBE Vapor Condensate (GMVC)”. The purpose of the study was to evaluate the chronic toxicity, potential carcinogenicity and exposure-concentration response relationships associated with inhalation of GMVC. Groups of 50 male and No response required.

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50 female F344 rats were exposed in H2000 whole-body inhalation chambers at GMVC vapor concentrations of 2g/m 3 (low level), and 20g/m 3 (high level) for 6 hours/day, 5 days a

week for 104 weeks.

The study was appropriately designed and adequately conducted to assess the chronic toxicity and carcinogenicity of GMVC. Protocol deviations noted did not adversely affect the outcome of the study.

Mean GMVC exposure concentrations were within a few percent of target through the study and were demonstrated to be reproducible from day to day. Relative percentages of major hydrocarbon components of condensate were constant throughout the exposure period. Homogeneity of vapors was distributed evenly within each chamber. Data on the daily percent normal usage demonstrated good agreement between anticipated and actual GMVC usage. Collectively, the GMVC whole body exposure procedures were properly conducted and allowed for the GMVC-induced toxicity and carcinogenicity assessments.

GMVC did not affect surv ival and no clinical observations of toxicity were attributed to GMVC exposure. Serological analyses on sentinels were negative throughout the study. (Note: data, in C-2/C-3, is for only 5 animals that were tested on 5-30-01 even though text states that 10 animals (5 male/5 female) are to be tested). Due to the staggered start of the two studies, sera was collected and analyzed for the 5 males (5/22/01) and 5 females (5/20/01) separately. No change

Text, p. 2-3, state that no contaminants were known to be in the diet or water in quantities that would be expected to interfere with study outcome; whereas it is stated that actual testing of water and diet were done annually, data is not presented. Diet and Water - the methods section has been

revised to indicate that th e diet was analyzed by

the manufacturers for various contaminants and

that established maximum allowable

concentrations were not exceeded. It will also indicate that water analyses were conducted routinely by an independent laboratory and that all analytes are sufficiently low as to not interfere with the outcome of the study. These data are now in Appendix D-1. 2-4; Appendix D1

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Body weight of high-level GMVC males and high and mid-level GMVC females were lower than control animals during the study, and at final sacrifice in high level GMVC

males and females. Text should note the percentage decrease in body weight at final sacrifice. Due to study start delay, the bodyweights of high-level males and mid and highlevel females at Day 1 were significantly below controls. Thus it would be appropriate for text/tables to present body weight gain data (this is routinely done) and may shed further light on the GMVC induced weight

losses. The percent decrease in body weights at final sacrifice have been added to abstract, summary and results text. The body weights and percent of treated:control body weights presented in Tables 3-5 and 3-6 are considered to adequately demonstrate the treatment effects on body weight, without addition of a table on body weight gains. i, S-1, 3-6, 3-7

Statistical differences in high level exposed males were noted in hematological parameters

at 12 months. Differences were not found in females, nor were they present at the 18

month and terminal s acrifice. Report correctly notes that these changes should be “interpreted with caution” . Report also notes that results in 12 month males possibly were affected “by the tail sampling techniques, which improved by 18 months” (p. 344). However, in the pathologist report (p. 0-5), it was noted that these results may result “from initial blood sampling difficulties”. Were there difficulties? If so, these should be

described in the results section to allow for full interpretation of these noted results. Additionally, it would be worthwhile to see and compare to similar data in the BGVC study; emphasis on the significance of these data would depend on the absence or presence of these findings within both studies. The difficulties in blood sampling at 12 months are explained on pg 3-15. A comparison of results between studies is difficult to interpret since there was difficulty in blood sampling in both studies at 12 mos, and there was no consistency in results between the two studies at the 12 month time point. Difficulties in drawing blood at 12 months (first time point) is considered to be a suitable explanation for the observed changes. No change

Brain weights, adrenal weights and epididymides weights were less than corresponding controls among various dosegroups. Absolute kidney and percent kidneyto-brain of all GMVC-exposed groups were significantly increased compared to controls. Kidney-to-body weights was significantly increased among males exposed to mid and high levels of GMVC. No response required.

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Histopathology revealed significant increases in proliferative lesions (adenomas/carcinomas) and nonproliferative lesions. Chronic nephropathy was observed at a high incidence in controls and exposed male and female study animals. Of particular relevance to the observed renal proliferation lesions, the severity of nephropathy was significantly increased in mid- and high-level males and high-level females. This finding may be a contributor to the induction of adenomas. Since severity, not incidence, may be a more relevant observation, conclusion section should emphasize this point more so than what is currently represented, including providing severity score in Table 3-16. Other non-proliferative lesions in GMVC-exposed rats were hyaline degeneration of olfactory epithelium (males) and respiratory epithelium (females) of the nose, an expected finding. Discussion of the severity of nephropathy has been added. Comparisons of effects seen with BGVC are also now provided. 3-21, 3-21, 3-23,

Table 3-13. For comparisons see Table 3-17, and pages 3-28, 3-29.

The most significant re sponses observed in this study were the statistical increases in

renal tumors and testicular interstitial

adenomas. Testicular adenoma incidence was quite high in control males and was significantly greater in mid-dosed males. A number of comments pertain to the renal tumor findings:

Text should more comprehensively note the renal tumor response and not just note the single statistical change in the mid-level male rats. For example, 10 tumors were observed in the study and all were found in exposed animals, and two carcinomas were observed in exposed animals (carcinomas are very rare in untreated rats). Renal tumor response have discussed in more detail for clarity and completeness. 3-17

Hyperplasias and atypical hyperplasias should

be evaluated and noted in tables and text where proliferative renal lesions are discussed; these lesions are considered relevant to the end-stage adenoma/ carcinoma

response. Hyperplasias and atypical hyperplasias were not observed in the kidneys of these animals. No change

It is surprising that (1) a statistical trend was Statistics have been performed on the combined Table 3-12, Pages

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not observed in the induction of renal lesions, and (2) that, in mid-level exposed rats, the renal tubule adenoma and carcinoma, combined, is not significantly statistical even though values are the sa me as adenoma alone. renal adenomas and carcinomas incidence data and added/discussed in the revised report. 3-17 and 3-18, Appendix O.

Text (p. S-1) states incorrectly that the

incidence of renal tubule adenomas was statistically increased in high-level males – it should be mid-level exposed rats. Also, text (p. 3-19; Table 3-14) has an error in reporting BGVC control results; the renal tubule adenoma, renal tubule carcinoma, combined must be “1(2%)” if the adenoma renal tubule

result is correct. Are BGVC statistical

differences noted? The text has been corrected to indicate that the mid-level exposed rats had statistically increased

renal tubule adenomas. The error in the

combined incidences of control renal adenomas plus carcinomas in Table 3-14 has been corrected. Statistics were not initially performed on the combined results; that has been corrected in the revised report. I, ii, S-1, S-2, Table 3-15, Appendix P,

Table P-2.

I recommend that a kidney step section review

is undertaken to further understand the unusual dose-response observed in renal tumors and to better assess the renal findings in the two studies (BGVC and MGVC) being compared. Following evaluation of chronic carcinogenicity studies (NTP and others), an

increased incidence of proliferative renal tubular lesions (hyperplasia, adenoma and carcinoma) may be observ ed in treated groups compared to controls. Often, full understanding requires further investigations of the kidney tissues to confirm the overall incidence of renal lesions. Kidney step

section reviews are conducted on additional sections per kidney. Results are summarized by treatment group which show the incidences seen in the original kidney section, the kidney section review, and a combined incident table of lesions seen in both the original kidney sections and in the step sections. Given the importance of the renal lesions following hydrocarbon exposure and the difficulties in evaluation, I also would recommend a Peer Working Group (PWG) on the kidney histopathology analys es for the MGVC and

BGVC studies. The Research Group questions the necessity of reconfirming the current study results and previously reported associations between gasoline and neat MTBE treatment and

induction of renal adenomas and carcinomas with a male kidney step section and/or Pathology Working Group review. Additionally, this type of analysis would not have the benefit of cell proliferation assessments and immunohistochemical staining for α 2-u globulin. Not applicable

Discussion should comment if earlier deathsand/or body weight losses noted in the highlevel GMVC exposed rats would affect the The results section has been revised to comment on the influence of mortality on renal adenoma/carcinoma response. 3-17

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renal adenoma/carcinoma response noted.

In previous studies in rodents, unleaded gasoline and MTBE have been postulated to

interfere with the hormonal milieu and potentially impact lesion development, including liver tumors and uterine atrophy in female mice, and the testicular and mammary gland findings in rats. In this light, it was interesting to note that data demonstrated increased mammary gland hyperplasia and

increased fibroadenoma in female rats. This

data should be noted in Results and discussed comprehensively. Testicular and mammary gland findings along with the pathologists conclusions have been brought into the main body of the report and pathologist’s report and discussed appropriately. A brief discussion on the mechanistic hypothesis of hormonal disruption has been added in the Results section. For mammary gland hyperplasia in males, there was a trend toward an increase in incidence, but because there was no hyperplasiain the mid dose group, there was no real doseresponse relationship. Further, if the hyperplasia were a result of GMVC exposure, tumors would have been expected, but were not seen in a concentration-dependent manner. With fibroadenomas in females, although the incidence of adenomas was statistically increased in the low dose (15%) and high dose (11%) groups, the control group incidence was extremely low (0%) compared to expected values reported in the literature by Boorman et al and Haseman et al. These ranges were 27- 40%. The references were cited by the pathologist in his report. 3-17 Table 3-12, P-12, P-13, Table

P-2.

The pathology report is presented in Appendix O. Page O-6 states that it is “unlikely that the (renal) carcinoma(s) was related to GMVC inhalation”; given the rate of occurrence of this lesion in untreated rats, the preponderance of literature findings and the other renal lesions (adenomas) seen in the present study, I disagree with this comment. The Research Group agrees that the renal

carcinomas were treatment-related for the same reasons given by the reviewer. Although the incidence of these lesions may not be statistically different in treated vs. control groups, the background incidence of renal carcinoma is low in male F344 rats, thus the observed increases in treated groups are biologically significant. Additionally, the lesions observed in this study confirm previously reported associations between gasoline and neat

MTBE treatment and induction of renal

carcinoma in male F344 rats. The discussion of these effects has been expanded and modified toconclude that the renal findings are treatmentrelated. i, iii, S-1, S-3, 3-17, 3-23, P-6, P14, P-19

The Pathology report notes the likelihood of “gross lesions sampling bias” (p O-8). Where findings are significant or possibly considered significant, or also seen in the BGVC study, then I would consider evaluating all animals This study was conducted in compliance with EPA OPPTS 870.4200 as specified in the Clean Air Act Section 211(b) regulation. Toxicology studies very commonly use protocols in which all tissues at the highest level of exposure are 3-20, Table 3-12

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in these dose groups to avoid the potential bias and difficulties in interpretations. As stated above, mammary gland hyperplasia and fibroadenomas of the mammary gland have a mechanistic underpinning to BGVC/MGVC exposure. Thus, I would like to know what information was used by the Pathologist to

lead him to choose to list both of these findings as “not attributable to GMVC exposure” (Table O-5 and O-6). examined for abnormalities, and at the lower levels of exposure, only target tissues and tissues with gross findings are examined further. While this might introduce what the report and the reviewer refer to as “sampling bias” and spurious incidences of st atistical significance at the lower exposure levels, the Research Groupbelieves the higher lesion incidences in the lowand/or mid-dose groups are more likely related to the low “n” based on evaluation of only spleens that appeared ab normal at necropsy at the terminal sacrifice. Had a significant number of findings been present in the high exposure group, all tissues in the lower dose groups would have been examined, as instructed in the protocol. This explanation for protocol-driven bias in the mid-dose groups is included in the report. The rationale for not attributing the mammary findings to treatment has been included, as described in the response to the

similar comment above.

211 (b) Research Group Comments 2

The following EPA comm ent made on BGVC report is also relevant to GMVC:

“Page O-6. 2/27 thyroid follicular cell carcinomas would be of concern for a tumor that is very rare in control rats, especially when associated with thyroid follicular hyperplasia. Likewise nasal squamous cell carcinoma is very rare in rats so should not have been so easily dismissed.” The RG feels the incidence data should be reviewed for both studies along with LRRI (and other?) historical control data to aid in interpretation of these findings. A table summarizing the incidence data from both

studies and NTP historical controls is attached

for LRRI’s convenience.

Squamous cell carcinoma was reviewed by the pathologist. The lesion was determined to be of oral, not nasal origin. This, along with historical data are now provided in the text and in the Pathologists report.

Without access to LRRI historical controls, the RG cannot form an opinion on the relationship between treatment and thyroid follicular cell

carcinoma incidence. Incidence from BGVC study is consistent with GMVC study. Thyroid tumors reviewed by the pathologist and found not to be statistically significant.

Comparisons of results in the BGVC and ii, S-2, 3-19, 3-29, Table 3-12; P7, P-15. Table P2

Comparisons found on pages 3-

23 to 3-30.

2 API minor editorial comments that were already addressed in th e 9/2/04 draft or were duplicative of EPA/independent peer review er comments are not included.

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GMVC report are now provided.

Relationship of GMVC treatment to testicular

interstitial cell adenomas: After review of data from both reports, and previous literature to include a recent review

on mechanisms of MTBE induced cancer in rodents (Cruzan et al., 2007 added to refs in report), rather than a definite association, an ‘equivocal’ association between treatment and tumor may be more appropriate. The high background incidence of this tumor (43/50 for GMVC and 48/50 for GBVC) tends to confound interpretation. Suggested revisions throughout the report. ii, S-2, Table 3-12, 3-17, 3-22, P7, P-15.

Page 2-1 . 1 st para, indicate that the concentration of the MTBE in the gasoline from which the vapor condensates were prepared was 15%. We are continuing to use 20% as that was the concentration in the vapor exposure atmosphere. I, S-1, page 3-24

and Table 3-14.

Page 3-3. Middle of page, change “Mortality” to “Survival Analysis” to be consistent with BGVC report. Revised to “survival analysis” 3-3

Page 3-18. 1 st sentence – indicate that the severity of the hyaline degeneration is very low (0.2-0.3 out of possible score of 4). Text has been revised to indicate range of severity scores. 3-22

Page O-5, O-7, O-13. Pathology report has what appear to be reporting errors for

• 12 mos male hematology changes

• “Increased incidence” of male rat nephropathy in the mid-dose group. See stat report, pg N-22: no differences in nephropathy incidence for any group were reported. Is the stat report in error? It does look as though there could be a

statistical difference between the control incidence of 44/50 (88%) and mid-dose incidence of 50/50 (100%) Text has been corrected to agree with hematology results table in body of the study (Table 3-11) and nephropathy incidence results in statistical report. P-5, P-9, P-17

Table P-3

Inclusion of comparisons between BGVC and

GMVC results in the conclusion section is Text has been revised to break out GMVC/BGVC comparison into a separate 3-23 to 3-30

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very useful. It is suggested that this comparison be broken out into a separate

section so as not to cause confusion with the conclusions from the GMVC study itself. section after GMVC study conclusions

Summary of the organ weight changes contradicts the summary tables in the results

section. Text corrected in ab stract and summary. i, S-1

Pg 3-1: Chamber Trials – the data for the chamber trials should be added to the report as an appendix, there is no appendix data to verify results summary provided in the text. The data are now provided in revised Appendix E. Results of feed and water analyses have been inserted as new Appendix D E-2 through E-8.

Pg 3-2: Homogeneity of Vapor Concentration – raw data should be added to report as an appendix to substantiate the summary

conclusions. The data are now provided in revised Appendix E. Results of feed and water analyses have been inserted as new Appendix D. E-9 through E-15.

1. 3 rd paragraph – last sentence stating that presence of atypical lymphocytes is consistent with age-related leukemia seemingly contradicts the finding in females of a significant decrease in atypical lymphocytes in high dose females, particularly since the splenic leukemia incidence was not significantly

different from controls. It should be noted that significance of the female finding is unknown (or some other explanation, if there is one). Text has been revised to indicate that the significance of increased incidence (over time) is unknown. There is no significant difference between the incidence of high dose and control females at the 24 month time point. 3-15

Pg 4-1,2: Protocol Deviations – need investigator conclusion as to the impact of deviation in “Temporary Loss of Environmental Monitoring System Operation” on the study (no impact?) Text revised to state deviations had no impact on study integrity. 4-6

Gross Findings at Necropsy – this data is not presented in summary form or by individual animal in either the report or the appendixes. This data should be included in the report (it is an OPPTS 870-4200 guideline requirement). Gross necropsy findings are now included in the pathology appendices M and N. M-2 and N-2

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211(b) Research Group QA/QC Reviewer

General Comments : Date of Site Visit and Review: November 8- 10, 2004

The draft final report for the Chronic Carcinogenicity Study was audited to assure compliance with the study protocol and GLP regulations (Part 79.60) and to verify that the report accurately reflects the data collected. One hundred percent of the text was verified with tables and appendices. At least 20 percent of individual values were verified and 20 percent of summary tables were verified

with individual data. No response required.

The following items require further

consideration:

1. Abstract, Page I, Fifth line: containing

“ approximately ” 15 %. Corrected to approximately 20% (sentence is describing the MTBE content of the vapor condensate, not the neat fuel used to make the condensate) I, S-1

2. Abstract, Page I, last paragraph, first line: This incidence is referring to the

mid-dose male rats and should indicate

so. Correction already made in 2 nd draft of report I, S-1

3. Abstract, Page viii: A subcontractor is

listed as the Statistician. This was not included in the protocol and should have been handled as an amendment, or now

as a deviation. This protocol deviation has been added 4-1

4. Abstract, Page viii: The Study Pathologist was changed from the original protocol and should have been

amended. This protocol deviation has been added 4-1

5. Abstract, Page x: The QA Statement should indicate more detail with respect to Phase inspections and data audits. What procedures or data were inspected/reviewed? More detail has been added.. xi- xv

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6. Abstract, Page xi, and the Introduction, page1-1: The GLP Compliance Statement should indicate that the study was performed in accordance with the EPA regulations........ It seems that there are many procedures, not necessarily indicated in the protocol, that needed to be conducted in compliance

with GLPs. EPA regulations and guidelines and exceptions

have been added. xvi

7. API will need to sign a compliance

statement as well. It will need to include

the fact that the method of fabrication was not available at the time the study initiated. It is, however, presently

available. This statement can be separate from the Lovelace one, or a signature block can be added with the above statement to the one presented in this report. Fabrication statement & signature block for API added to existing compliance statement xvi

8. Page 2-3, last sentence of first paragraph: Oxygen concentration was

monitored but not recorded. It was recorded at some interval, wasn’t it? Please clarify. Oxygen concentration was monitored but not

recorded.

9. Page 2-4, Table 2-1, last line: All protocol-required tissues collected. Text revised. 2-5

10. Page 2-11, Statistics: For Body and Organ Weights there were different

statistical methods to be used. The ones listed here are for Organ Weights. Please indicate how Body Weights were

evaluated. The text was revised to indicate that the statistical methods for body weights, organ weights, organ to body weight and organ to brain weight calculations all were done using the

same statistical methods. 2-12

11. Page 2-12, Analysis of White Blood Cell

Total and Differential Cell Counts: The protocol-prescribed statistical methods were not used, probably, because

distributions were skewed. Since there were significant interactions between time and group, comparisons were done This protocol deviation has been added. 4-2, 4-3

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to compare dose groups at each time point and to compare time points within each dose group. Th e protocol did not indicate that the three time points would be compared. Please comment.

12. Page 2-13, Histopathology: The protocol indicates that statistical evaluation listed as number 2 (logistic regression) would be used, as well as the Hoel-Walburg test. The other tests (1, Cochran-Armitage and 3, Fisher’s exact) may be used as well, but the logistic regression and Hoel-Walburg test were protocol-required. Please comment. This protocol deviation has been added. 4-2, 4-3

13. Page 3-1. Table 3-1: I could not verify exactly the values reported for the Day 2

and 3 Achieved Concentrations. Could this be a rounding issue? Please verify

these values. The data were verified against the date in Notebook 7401. The day 2 and Day 3 achieved concentration stability are correct. They are also correct in Appendix E (formerly Appendix D). No change

14. Page 3-3, Chromatographic Profiles of GMVC: The reason for analyzing all large tanks of GMVC was to show that all tanks were similar in profile and to

show that the test material was stable throughout the entire testing period.

This should be indicated. This explanation was added to pg 2-1. 2-1

15. Page 3-3, Animal Disposition: The second sentence should probably read, “The majority of animals not reaching

termination underwent moribund

sacrifices.” Revision made. 3-5

16. Page 3-4, Figure 3-1: This is not the same figure as in the Stat. Report of the

same information. Which one is correct? The figures now match. 3-4

17. Page 3-6, Table 3-5, the Mean Body Weight for Day 7 for Group 2 should be 193.8, I believe. As well, why are some values carried out to a tenth of a gram, This has been corrected. 3-7, 3-7

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while others are not? Please clarify.

18. Page 3-9, second sentence: Body weights of high-level male and female rats

remained statistically significantly below control values for the study. Text has been revised. 3-9, last paragraph.

19. Page 3-9, Table 3-7, footnote b is not on

the table. Should it be? There is no footnote b 3-7

20. Page 3-10, third to last sentence: The epididymides weight was significantly decreased among mid -level males. As well, liver organ-to-body-weight ratios for high-dose males are statistically significantly increased. Text revised for epididymides.

LRRI to verify if liver organ-to-body weight significantly increased. This is not indicated in table 3-9, nor do the values appear to be different by visual inspection.

The liver statistics were verified on Jan 29, 2009 by JB. The liver to body weight ratio for high

level males at final sacrifice was not significantly increased. No text change

necessary. 3-10

21. Page 3-11 to 3-13: these tables are only terminal sacrifice animal organ weights. This should be indicated clearly on the

table. Table titles now indicate the weights are for

terminal sacrifice animals. 3-11, 3-13, 3-14.

22. Page 3-10: It would probably be easier to refer to page numbers as Appendices J-2 through 6 are not clearly marked (only marked with page numbers J-#). References to the appendices are now included. 3-10, 3-12.

23. Page 3-12: The si gnificances noted for Brain, group 4 male, and Kidney, group 4 male are significant at the P< 0.01 level, not 0.05 as indicated. No change. Significance levels were set at

p< 0.05. 3-10, 3-12.

24. If one compares the last clinical observation to the necropsy observations, it appears that the last observation of “skin growth” frequently

was neither confirmed nor denied. One In life clinical observations recorded in Path Tox did not necessarily follo w the same format as recording gross lesions at necropsy on IANR

forms. In the former cas e, examples of Path/Tox

notation of skin lesions include “Hair/skin/skin No page.

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can only assume that the skin growths noted clinically were in fact preputial masses or enlargements. Please

comment. growth (small), Hair/skin/sore (superficial wound/lesion)”, with no notation as to location. In the latter case, at necropsy more precise

notation of anatomical location and more current notation of size occurred. Thus, correlating in life and necropsy observations may be difficult, requiring assumptions as to the location of the skin growths. These growths were likely subcutaneous masses, preputial or otherwise.

25. Page 3-14, second paragraph, second sentence: In all cases, values for the high-level group males were greater. Corrected. 3-15

26. Page 3-14, second paragraph, fourth

sentence: The differences were not really present in males at the 18 or 24 month intervals. It may also be helpful to indicate what the problem was with sampling the blood at the 12 month

interval. Text was revised to indicate we had blood sampling problems. 3-15

27. Page 3-14, last paragraph, second

sentence: testicular interstitial cell

adenomas..... Text has been substantially revised in this

section and the word interstitial has been

included. 3-17

28. Page 3-16: When discussing the statistically significant incidence of Leukemia in the mid-doses, it is true that the percentage of those examined is misleading, but with the frequency of the lesion equal to that seen in the high-dose group, one can only assume that it would go up if all spleen tissue were examined. At what incidence would significance

start? An additional header has been added before discussion of leukemia and mammary gland

“Proliferative Lesions Not Attributable to GMVC Exposure”. Leukemia and mammary gland fibroadenoma are now discussed under the

above header. 3-20

29. Page 4-1, Protocol Deviations,

Environmental Conditions: These excursions from protocol-required ranges need to be further indicated. Some sense of how long and to what

extent these excursions occurred need to be given. As well, the periods of data loss were sometimes days (May 24-28, This protocol deviation and several others have

been added. 4-5

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02 and July 11-15/02 when the RH meter was not working). Give lengths of malfunctions and specific reasons why

these excursions and losses did not affect the integrity of the data.

30. Page 4-1, Protocol Deviations: There was also a day on which the airflow dropped affecting the next day’s exposures (11/28/01). Exposures were not run that day. This should be

indicated as a deviation. This protocol deviation was added. 4-10

31. Appendix B: Test Material

Characterization for Ta nk A-27 (done on 7-8-02) was not reported. Should it be? This data was added as pg B-21. B-21

32. Appendix B, Tank 16: It is indicated that this tank was not analyzed. It was (in effect) analyzed as part of the weekly analysis of the atmosphere. This should be noted as a deviation and the analytical data applicable to this tank (weeks used for exposures) should be indicated. This protocol deviation was added. 4-3

33. Appendix D, page 3, high dose value,

7/6/01: Should this value be 20.53? Please clarify. The correct value is 20.53. The number in the table was corrected in Appendix E. E-33

34. Appendix D, page 4: The standard deviation for the high dose group exposures for 9/17/01 now appears 0.11,

but in all other audits the value was 0.54. How could this have changed? The value of 0.54 was an error not noted in previous audits. The value in the report (0.11) is

correct and was verified to the 9/17/01 raw data

sheet. E-34

35. Appendix D, page 6: The Actual usage value for 4/1/02. Please verify that 5320

is correct. I find 5515. The value 5320 is correct and verified to the raw

data sheet for 4/1/02. E-36

36. Appendix D, page 6: The Actual usage value for 4/1/02. Please verify that 5320

is correct. I find 5515. Repeat comment, see number 35. NA

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37. Appendix D, page 21: The heading of

Table D-2 is unclear. These are not the components of GMVC, there is no

MTBE. How are these values relative to

BGVC? The correct table is now included. E-51

38. Appendix D, page 23: The Table D-3, Day 2 and 3: Values could not be

verified. Are these values of the reperformed stability tests? Please verify

these values. The stability tests were performed on three consecutive days. They were not re-performed.

The values are correct and re-verified to

Notebook 7401. E-53

39. Appendix D, page 24, second distribution assay: The daily exposure

sheet indicates that data are in Notebook 7401. I could not find any data in

Notebook 7401 that was recorded on

6/6/01. The daily exposure sheet for 6/6/01 has a note

that the information about the control chamber valves (not values) was to be found in Notebook 7401. The protocol-required chamber

distribution data for the chambers with animals present were entered on a “chamber homogeneity data sheet” located in the study file. The control chamber was tested on 6/6/01;the low level chamber on 6/8/01 and the highlevel chamber on 6/13/01. The data in Appendix D (now E) were re-verified and are correct. E-54

40. Appendix E, Page 4: Analytical data for week of 7/23 and 9/10/01 are missing from the report. There is no mention of

this in the text or in the deviations section of the report. There should be. Please include the reason for missing data as well as any affect it could have had on the study. Appendix E, Page 4: Analytical data for week of 7/23 and 9/10/01 are missing from the report.

There is no mention of this in the text or in the deviations section of the report.

There should be. Please include the reason for missing data as well as any affect it could have had on the study. These deviations and statement of impact are included in the report. 4-3

41. Appendix E, Page 9: Analytical data is

labeled as 5/17/01 in the middle of Dec. This is actually data collected for the

week of 12/17/01. The date on the table and was corrected in the second draft of the report. (re-verified by JB

2/1/09 F-9

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42. Appendix K, Page 16, Animal number 470: The monocyte count should be 1 and the eosinophil should be 4. The monocyte count of 1 and eosinophil count

of 4 are correct. The values were corrected in

the 2 nd draft of the report. JB verified that they

were corrected on 2/1/09. L-16

43. Appendix K, Page 22, Animal number 770: There is 1 NRBC present for this animal, not 0. The value was corrected to 1 NRBC in the second draft of the report. The change was verified by JB on 2/1/09. L-22

44. Appendix L, Page 257, Animal number 410: Gross necropsy observation does not indicate “pituitary enlarged” but the gross to micro report does. The gross necropsy observation indicates “discolored pituitary, but the gross to micro does not. Please clarify. The pituitary for animal 410 was discolored, not enlarged. The entry was edited in Path Tox N module during preparation of the 2 nd draft of the

report. M-274

45. Appendix L, Page 256, Animal number 406: The gross observation “tooth perforation, maxillary was not entered on gross to micro. This lesion was not trimmed, no section was made, and no correlating entry was made. The lesion related to tooth perforation was actually trimmed in as a portion of the nasal

turbinate. The correla tion was entered by the pathologist. M-273

46. Appendix L, Pate 256, Animal number 406: The gross observation “tooth perforation, maxillary was not entered on gross to micro. This lesion was not trimmed, no section was made, and no correlating entry was made. Repeat comment. See number 45. M-273

47. Appendix M, Appendix L: At the end of each of these appendices, there is a Lesion incidence summary with K-S (Kolmogorov-Smirnov) comparison. It is somewhat misleading in that there Use of the Kolmogorov-Smirnov statistic in Path Tox to assess the statistical significance of the severity of nephropathy was described in the

Statistics section of the 2 nd draft of the text. A protocol deviation is also included. 2-14, 4-2.

48. Appendix O, page 3, Histology: Please give more identifying information about the Path-Tox database including version numbers, amendments, etc. The information has been added. P-4 to P-5.

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49. Appendix O, page 5, next to last

sentence: The increase in all groups in atypical lymphocytes...... Text now indicates high dose and control rats. Comparisons are made only between the two

groups. P-5

50. Appendix O, page 6, third paragraph,

third sentence: One cannot confirm that

most testicular adenomas were bilateral and multiple as they were not reported this way. This description has been removed. P-7

51. Appendix O, page 6, third paragraph, last sentence: It appears that this sentence is not true. For example, Animal numbers 718, 650, and 644 have reported both interstitial cell hyperplasia

as well as bilateral adenomas of the testes. Please clarify. The text has been modified to say that interstitial cell hyperplasia was recorded PRIMARILY in

testes without tumors P-7, paragraph 2

52. Appendix O, page 7, Table O-3: The kidney nephropathy incidence in the

mid-dose is indicated in the text as statistically significant. Should this be

indicated here? The incidence of chronic nephropathy is not statistically increased and is not noted as such in

either the text table 3-13 or in Table P – 3. The severity score is increased at both the mid and high levels, and this is correctly noted in the

tables and text. 3-20, Table 3-13, P-9, Table P-3.

53. Appendix O, page 8, Table O-4: The Nose/Turbinate 2 findings are also statistically significantly positive for trend according to the text. Please clarify. Table corrected. P-4

54. Appendix O, page 10, Table 0-6, Pituitary Gland: There is a sampling

bias in the statisti cal evaluation of the pituitary gland lesions because only gross lesions of the pituitary were examined, but since the incidence is already as high as in the high dose, further examination of tissue could only increase the incidence of the finding. It

is not clear what one can draw from

these results. LRRI agrees. There is a high incidence among controls, and no clear dose response with

exposure. Table P-6

55. There is no gro ss observation summary Gross observations have been included in the M-2, N-2.

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table. pathology appendices.

56. Gross to Microscopic correlation: Animal number 713, liver foci “1-4”

foci. Should be 1-4 mm. The IANR form for animal 713 was rechecked.

The form showed that the liver had 1 – 4 foci.

The size was not measured. No revision is

necessary. No change

57. I do not have responses to QA audits for the following dates: Reports dated 10/23-24/01, 1/28-29/02, 5/20-21/02,

9/24-25/02. Responses have been incorporated into the revisions of the final report. The final report has been reviewed by Christine Sexsmith.