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EPA

At your request, NHEERL 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

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. See responses to extramural peer reviewers’ comments

below.

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

EPA Comments on Study FY01-027

BGVC

Page 3-17. The nephropathy present in

the male rats is consistent with whatwould have been expected from a2uglobulin overload. Report revised to state that increased severity of CPN in

male rats is consistent with a2u-globulin overload. ii; S-2; 3-22; P-13, P14.

Page 3-18. Is accumulation of the globular eosinophilic material in the Diagnosis of nasal respiratory and olfactory changes was No changes

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 numbers will need to be corrected to the final page numbers. Also note, page designation of text revision in response to comments was specifically requested by EPA.

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respiratory and olfactory epithelium considered a degenerative change? In a series of studies by Lewis et al., these globules were metabolizing enzyme

accumulation in dilated SER after exposure to various chemicals (Nasal Toxicity and Dosimetry of Inhaled Xenobiotics; Fred Miller ed. Taylor and Francis 1995). based on H&E slides, only.

In the absence of definitive

tests to determine the composition of the eosinophilic material accumulations, these changes

were classified as degenerative.

Appendix O. All 50 animals are always

examined and included in the tables

however less than 57% survived to greater than 600 days. Should all 50 animals have been used consistently as the denominator in all summary tables for all dose groups? 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, 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. No changes

Page O-4 and 5 (comments also relevant for O-8). 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 typica lly associated with The RG and investigators agree with EPA on all points.

The referenced section in the pathologist’s report, and corresponding sections in the results section in the body of the report have been revised to note the points outlined ii; S-2; 3-22; P13, P-14

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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 that rat ages, usually after 1-year of age and so would not be expected to be present in the kidney of a 2-year old rats. Any eosinophilic droplets present in the female rat kidney were likely due to nonspecific protein resorption by the proximal tubules. here by EPA.

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.

RG comments to LRRI in Draft 2 (7/20/07) LRRI pathologists to review incidence

data to address this EPA comment.

The RG feels the inci dence 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.

Based upon the RG review of the attached table, it appears that the nasal squamous cell carcinoma may be treatment-related, particularly when

evaluated as nasal turbinate levels L2 – L4 combined. Incidence from GMVC study is consistent with BGVC study.

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 GMVC study is consistent with BGVC study. Statistical analyses of thyroid

adenomas and carcinomas

combined showed no statistical significance.

Historical data from one LRRI bioassay conducted in the early 1990’s showed the following incidences of Thyroid gland proliferative changes in male rats (values are for control, low, mid and high dose material). In no case were changes significant

or considered treatment

related.

C Cell adenoma, 13%, 17%,

8% and 13% C-Cell Carcinoma: 2%, 3%,

0% and 0%. Follicular Cell adenoma, 0%, 0%, 3% and 2%

There were no follicular cell

carcinomas.

With regard to squamous cell carcinoma in the nose,

tumors identified in L2 - L4

cannot be combined because the same tumor penetrated all sampling sites. The tumors found were not distinctly unique entities. iv; S-4; 3-21, 3-22, 3-27; Table 3-12; P-5 thru P-12; P-19,20; Table P-1

Thyroid tumors are discussed on pages 3- 21 to 3-22 and Table 3-12. P-9 and P-10,

Table P-1.

Nasal squamous cell carcinoma isdiscussed on page 3-19 and Table 3-12. P7, P-8, Table P-1.

Testicular mesothelioma is discussed on page 3-20. P-8, P-9.

Overall conclusions regarding these

tumors are:

“Consistent with previous studies and the concurrent GMVC study, chronic exposure to BGVC did enhance the development of renal adenomas and

carcinomas in male rats. In contrast with the concurrent GMVC chronic study, BGVC may also have enhanced squamous cell carcinoma in the nasal specimens, testicular mesothelioma, and thyroid

follicular carcinoma. Consequently, due primarily to treatment-related effects in the male kidney, nose, testes, and thyroid,

chronic inhalation of Baseline Gasoline Vapor Condensate was determined to be carcinogenic in male rats in this study.

Chronic inhalation of Baseline Gasoline Vapor Condensate was determined not to be carcinogenic in female rats in this study.”

Comparisons with GMVC are made in that

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report.

Table O-1. There seems to be no difference in severity of nephropathy in male rats. We agree with Tom Goldsworthy, it makes one wonder why

there were no renal tumors after exposure to UG.

Is there an explanation? There were statistically significant increases in combined incidence/severity of nephropathy in the male mid- and high-dose groups compared to controls. The

incidence of male renal tubule adenomas (1/50, 1/50, 4/50, 0/50 for control, low, mid and high dose groups, respectively) was increased in the mid-dose group. The

incidence of male renal tubule carcinomas (0/50, 0/50, 3/50, 0/50 for control, low, mid and high dose groups, respectively) was

also increased in the middose group. These results were reported in Appendix M and Appendix P.

These effects have been

determined to be treatmentrelated.

These results have been added to, and discussed in the pathology report (Appendix P) and main body of the
report. ii; S-2; 3-22; 3-23, Table 3-12 & 3-13; P13, P-14 Tables P-1 & P-2

The authors of this work may wish to review the following paper for

additional information:

Standeven and Goldsworthy. J Toxicol Environ Health 43:213-224, 1994. They found that whole UG and heavy UG caused comparable increased in PROD, EROD and hepatic LI mice. Light UG caused relatively small increased in PROD, EROD and not change in LI in mice. The RG and investigators have reviewed this paper. In light of the fact that (1) liver tumors from UG were only observed in female mice, (2) the referenced study was conducted in mice, not rats, and (3) in the current study

no liver tumors were

observed in female rats and only a low, non-significant number of hepatocellular adenomas were reported in male rats (0/50, 1/38, 1/37, and 1/50), it was decided not No change

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to include discussion of this paper in the report.

Peer Reviewer – Schlesinger

I have reviewed the above captioned report. The conclusions are generally

sound and there were no deviations from protocol that would have affected the

results. No response required. NA

Since this is a carcinogenicity study, the summary and conclusions should clearly

indicate the effect or l ack of effect of the test agent on carcinogenesis. As currently written, one has to read

“between the lines”. The text in the Abstract, Summary and in section 3

has been revised to be more specific regarding the carcinogenicity of BGVC. Specifically, the conclusions

are that BGVC is carcinogenic in male rats due

to effects on renal

adenomas/carcinomas combined, nasal squamous cell carcinoma, thyroid, and

testicular mesothelioma. BGVC was not carcinogenic

in female rats.

We have removed reference to BGVC being an irritant

because there is no

documentation in the

literature to substantiate the specific claim of irritancy versus toxicity with respect to observed degeneration of nasal respiratory epithelium. iv, v, S-4, S-5, 3-27

p. 2-11. Statistics. The Dunnett’s test is generally a post hoc test to evaluate differences between test agent and control group. What test was used to determine if there were any differences between the groups prior to use of the post hoc test? Bartlett’s test was used to assess the homogeneity of the

data. An ANOVA is run.

Dunnett's or modified t tests are used to assess group

differences if a trend is detected by the ANOVA.

The text has been revised to

include the ANOVA. The text

has been modified for

accuracy. 2-13

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p. 2-11. Histopathology. The description of the statistical analysis is somewhat confusing. The investigators

should rewrite this section so as to better clarify justification for the different test being employed. After review of section indicated, it was decided that the description of statistical methods was adequate once

clarification of the use of the Kolmogorov-Smirnov statistic by the pathologist was provided. 2-14

p. 3-1. Homogeneity of Vapor Concentration. Values are presented for spatial variation, but it is not clear what these are percentages of. Spatial variation is the percent difference in

concentration between the eight sampling locations in

the front and back of the chamber compared to the

concentration at the reference position in the middle of the

chamber. This has been

clarified in the text. 3-1

p. 3.2. Confirmation of Absence of Aerosol. Were any measures for particles taken during the exposures?

What was the lower limit of detection for the particle analyzer and what size range did it measure? These measurements were taken during the chamber

trials. No measurements for particles were taken during the actual exposures since

animal dander could contribute to particles in the exposure atmosphere. Details

on the aerosol detection methods, limits of detection and measurement size range have added to the report

results and the aerosol scientist report (Appendix E).

Note – LRRI aerosol scientist will need to provide this information & revisions, since the information is not currently presented in Aerosol Chemist Report in Appendix E – (page 170 by Adobe page count). Section on Absence of AerosolConfirmation is now properly labeled. E28.

A TSI Aerodynamic Particle Sizer (model 3321, TSI, Inc., St. Paul, MN) was used.

E-69.

Efforts were made to identify limits of detection and particle size range, but no

definite information was obtained.

p. 3-2. Homogeneity of Vapor

Concentration. Values are presented for Spatial variation is the percent difference in 3-1

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spatial variation, but it is not clear what these are percentages of. concentration between the eight sampling locations in

the front and back of the chamber compared to the

concentration at the reference position in the middle of the

chamber. This definition has

been added.

p. 3-3. Survival Analysis

. The last paragraph on this page is unclear. What is meant by day of 25% mortality and what is the significance of this extrapolated number? Day of 25% mortality is the day of the study at which

25% of the animals had died. Analysis of this parameter among the four treatment groups is conducted to

determine if survival was affected by treatment.

The day to 25% mortality was calculated using the Kaplan-Meier survival analysis using PROC

LIFETEST in SAS. This is

now clarified in the Methods

section on Statistics/Survival Analysis. 2-13, 3-3

p. 3-6. Body Weight Gain. It is noted that body weights of males in the mid and high level BGVC exposure groups were significantly below control values throughout the study. This is not evident from the data presented. Differences seem to appear sometime after day 95. Tables 3-5 (males) and 3-6 (females) show in italics the group differences in body weight from control (see footnote to these tables). The

italicized text has now been

bolded to make the table

easier to read. There also appears to be some confusion by the reviewer between male and female body weight data;

decreases in mid-dose female body weight appeared after day 95. The text has been revised to clarify body weight gain results. 3-6, 3-7, 3-8

p. 3-6. Absolute Organ Weights at Final Sacrifice. Does the investigator consider the statistically significant changes as also biologically significant since there Statistically different values in absolute organ weight are not necessarily biologically significant. Mean kidney 3-6

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is not always an apparent trend? weights in exposed females were all greater than the corresponding control values,

but were not increased in a concentration-dependent

manner. The incidence of chronic nephropathy in

females did not correlate with kidney weight. The text has

been revised to indicate that these changes were not

considered treatment-related.

p. 3-12 Percent Organ to Body Weight

at Final Sacrifice. The same comment as in #9 above applies here as well.

Copied conclusion sentence from organ:body wt. section to organ:brain wt. section in report, since LRRI’s conclusion applies for both parameters,

i.e . no exposure response. – report pg 3-

16 The statistical differences in percent organ to body weight are unlikely to be biologically significant. Many of the significant differences were larger organ:body weight values in treated groups, which is likely an artifact of reduced bodyweights in treated groups. The text now

focuses on lack of an

exposure response relationship between organ to body weight ratios and exposure concentration, leading to the conclusion that the effect is not exposure

related. 3-12

p. 3-12. Terminal Body Weight. An explanation of why interpretation of the data using animals euthanized over several months is more complicated is needed. If the investigators knew this would be a problem prior to the start of the study then why was a study design used that may have complicated statistical analysis of results? Evaluation of terminal body and organ weights were

conducted to determine if there were changes that could be related to lesions (e.g. nephropathy correlated with kidney weight/ testicular

tumors with increased testes weight). These weights were included in the report because they were recorded. Since

cause of death of euthanized animals was not determined, the significance of the changes that did not correlate

with terminal sacrifice data is

unknown. The text has been 3-12, 3-15

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revised to state this.

p. 3-16. Table 3-11. It seems misleading to indicate a significant trend

over time when values at 12 and 18 mo are essentially zero, e.g., with atypical lymphs and also absolute blasts. The statistics show a trend,

and there is an increase in values with time, even if the average remains essentially

zero. No change to Table 3-11

p. 3-15. Hematology. Would additional information have been provided if interaction between time and exposure was statistically evaluated?

In what way did the blood sampling technique affect the ability to ascertain differences between the exposure groups, and why was such sampling technique used in the first place? As stated in the first paragraph on page 3-15, because changes in hematology parameters over time were expected due to aging and disease, comparisons between groups at each time point likely provide more useful information than comparing

across time. It is difficult to say whether additional useful

information would have been obtained if time and exposure

interactions were evaluated. At the 12 month sampling interval, technicians had difficulty drawing tail vein blood for hematology analysis. No change, page 3-15

Peer Reviewer – Goldsworthy

The study was appropriately designed and adequately conducted to assess the chronic toxicity and carcinogenicity of

BGVC. Protocol deviations noted did not adversely affect the outcome of the study. On page B-13, it was noted that tank BG22 was not analyzed; I would have thought this would have resulted in a deviation. Mean BGVC exposure concentrations were within two percent of target throughout the study and were demonstrated to be reproducible from day to day. Relative percentages of major hydrocarbon components of condensate were relatively constant throughout the exposure period. A protocol deviation to report the missed analysis for tank

BG22 was noted in the

materials and methods and added as a protocol deviation. 2-2; 4-5

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It is not clear why two batches of BGVC were used in this study. As shown in Appendix B, test article characterization

was different in the second batch ascompared to the first batch (see B-1, B18). Increased levels were observed in N-butane, 2, 3-dimthylpentane, 2- methylpentane and 3-methylpentane; decreases in N-pentane and N-hexane.

These differences were borne out in the weekly gas chromatographic profiles of chamber atmospheres Appendix E; e.g. E-24, E-25.

Text should note these differences and

comment as to the relevance or lack

thereof of these differences in the batches used in the study. It was necessary to prepare a
second batch of test material because the first attempt to conduct this study was

aborted. The second batch of

test material was used for the last 12-13 weeks of the study.

While there were differences

in the relative concentrations

of various alkanes and isoalkane components, the Workgroup does not feel that

these differences influenced the outcome of the study.

This has been addressed in the text of the revised report.

This statement has now been added to text – an oversight

in last draft. 2-1

Homogeneity of vapors was distributed evenly within each chamber. Data on the daily percent normal usage demonstrated good agreement between anticipated and actual BGVC usage. Collectively, the BGVC whole body exposure procedures appear to be properly conducted and allow for the BGVC induced toxicity and carcinogenicity assessments. BGVC did

not affect survival. No clinical observations of toxicity were attributed to BGVC exposure. Serological analyses on sentinels were negative throughout study. No response required. NA

Text states that no contaminants were known to be in the diet (or water) in quantities that would be expected to interfere with study outcome. Data is not presented. Whereas environmental conditions appeared not to effect animal health or study data, the humidity readings, especially in low dose animals, were unusually low (20-40% range) for The methods section has been

revised to indicate that the diet was analyzed by the

manufacturers for various

contaminants and that

established maximum

allowable concentrations

were not exceeded. It also indicates that water analyses 2-5

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several months. were conducted routinely by an independent laboratory and that all analytes are sufficiently low as to not

interfere with the outcome of the study. Results of feed and water analyses are now provided in Appendix D.

Body weights of males in mid and high exposure levels were below control values throughout the study; some but less consistent body weight decreases

were observed in treated female rats. Body weights of high-level males and mid and high level female rats were

below control values at terminal

sacrifice. A number of absolute or relative organ weight differences were noted in treated animals. Interestingly increased kidney weights were seen in BGVC treated groups in moribund rats

but not in terminal sacrificed rats. No response required. NA

I suggest re-stating the hematologic data in the abstract and summary. As currently stated, total and differential

white blood cell coun ts are viewed as not attributable to BGVC exposure. However, a number of statistical changes were noted (less emphasis is placed on these findings since they were not at all exposure levels or all time points). Additionally, the increase levels of MCL may have affected the WBC

counts observed in treated animals. Lastly, it is interesting that the GMVC study also demonstrated WBC changes predominately at the 12 month time point. Collectively, the results, conclusions and summary sections would be more accurate to explicitly state the findings noted and to phrase the findings as “unknown” when considering if they are attributable to BGVC exposure. The text currently indicates

that there were no effects on

WBC that could be attributed to BGVC exposure. Comparing groups at each time point reveals only one significant difference at 24 months (absolute lymphocytes). There were

several differences in females at 12 months, but these could be related to sampling difficulties (as discussed before). The conclusions and summary sections have been

revised to indicate that it is unknown whether changes might be attributable to BGVC exposure. ii; S-2, 3-15; P-20; P-21

BGVC exposure increased nephropathy The Workgroup agrees that 3-22, 3-23, P-13, P-14

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in both male and female rats, with greatest severity, as expected in male rats. Pathologist reports the apparent lack of classical 2u-globulin changes; this finding would not be expected in aged animals. BGVC exposure also caused degeneration of nasal epithelial degeneration. age-related chronic progressive nephropathy masks the classic signs of light hydrocarbon-induced nephropathy in male rats after approximately one year. The

text has been modified to
present a more comprehensive discussion of

these results.

Pathology report (O-3) notes that ‘a few

tissues’ were taken and examined that were not stated in the protocol; how and why were these ‘extra’ tissues selected

for inclusion? Because of anatomical juxtaposition, sometimes non-protocol required tissues were also sampled along with protocol required tissues. In order not to compromise thequality of the protocolrequired tissues, the nonrequired tissue was not removed during trimming. Iftrimmed and on slide, nonprotocol required tissues were read. Specific examples related to this study included salivary gland (taken

sometimes with mandibular lymph node) and thyroid gland (attached to trachea and esophagus). P-3

Both summary and abstract are incorrect in noting that ‘exposure to BGVC did not result in the development of hyperplastic or neoplastic lesions in

male of female rats”. Several statistically significant increases were noted, including the MCL mesotheliomas (in numerous tissues), keratoacanthomas, thyroid follicular cell carcinomas, and squamous cell carcinoma in the nasal passages. These responses were significant by the Fisher’s exact test, an appropriate analysis when survival among test group is similar. Many of these responses may not be attributable to BGVC exposure due to the lack of dose responses can be

‘dismissed’ with cert ainty e.g. there may Incidence data for hyperplastic and neoplastic lesions where significant

differences were observed

has been added to the results section of the report. The abstract, summary, results, conclusions, and pathologist report sections have been

revised to include more discussion about potential

associations between the

lesions mentioned and BGVC exposure. This is especially true for nasal squamous cell carcinoma (only level 3), mesothelioma in testes, and thyroid follicular cell iii - v; S-3 – S-5; 3-23 through 3 -27; Table 3-12; P-5 through P-22

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be a relationship with the nasal histopathology degeneration and the induction of squamous cell carcinoma in the nasal cavity. Reports, including abstract and summary, should note these findings and discuss them appropriately. carcinoma.

I cannot determine if or where in the report the statistical analysis of “renal adenomas and carcinomas, combined” exists. Given the tumor profile in the renal data set, this may be an important endpoint to assess. Statistics have now been performed on combined renal

adenomas and carcinomas. ii; S-2; 3-17; Table 3-12 P-5; Table P-1, Appendix O

The most unexpected result of the study was the lack of a tumor response in the male rat kidney of exposed groups. An increased renal tumor response would be expected based on historical data as well as the concurrent GMVC study. The report itself is incomplete in addressing and reporting this critical endpoint. The Pathology report does not mention the renal lesions whatsoever, despite this being the number one target organ of

interest. There were no renal adenomas, carcinomas, or

mesotheliomas in females.

There were 3/50 male rats in the mid dose group with renal carcinoma, and 0/50 in the

control and other dose groups. The incidence of renal adenomas was 1/50, 1/50, 4/50 and 0/50 for the control, low, mid and high male dose groups respectively. Minimal renal hyperplasia was reported in males (1/50 in high dose males only). While

incidences of renal adenoma or carcinoma taken separately were not significantly increased in males, there was a statistically significant

increase in the combined

incidences of renal adenomas

and carcinomas in males

which is considered to be

treatment-related. This is now discussed in the text abstract, summary, results section as well as in the pathologists report. The statistician report

reflects data on statistics of

combined incidences of renal and thyroid adenomas and

carcinomas. ii; S-2; 3-17; Table 3-12; P-5; Table P-1 Appendix O

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Concern exists in utilizing two different pathologists to analyze the GMVC and BGVC studies, limiting their comparative nature.

Data sets within and between these two studies indicate a potential tumor response to BGVC, BGVC plus MTBE, or both. Hyperplasia and especially atypical hyperplasias are useful information to support and evaluate equivocal renal tumor responses and

should be assessed. With respect to the concern about different pathologists examining the renal lesions,

there was considerable

interaction between the two pathologists to ensure consistency in the evaluations. One pathologist read the gasoline/MTBE carcinogenesis bioassay study, while a second pathologist read both the 90 day toxicity study (the first BGVC study with doses unacceptably higher than target was aborted and carried out to 90 days) and the 2-year carcinogenesis bioassay of BGVC exposure. During the 90 day study, both pathologists reviewed lesions evident within the kidneys of the BGVC exposed rats and discussed chronic progressive nephropathy and hydrocarbon-induced nephropathy of rats and what might constitute differential findings among these entities. With this background, both pathologists proceeded to

read each of the carcinogenesis assays, and

further discussions on chronic progressive nephropathy of rats, hydrocarbon-induced nephropathy of rats, and renal hyperplasia/neoplasia took place. Select lesions from the carcinogenesis assays as well as the studies' reports were reviewed by both pathologists. No change required.

Furthermore, I recommend that a kidney step section review be undertaken to further understand the response observed

in the BGVC induced renal tumors. The Research Group questions the necessity of reconfirming the current study results and previously No change

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Following evaluation of chronic carcinogenicity studies, an increased incidence or suggestive findings of an

increased incidence of proliferative renal lesions (hyperplasias, adenomas, carcinomas) may be observed in treated animals as compared to control values. Often, full understanding and correct data interpretation 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; this allows for a much greater comprehensive evaluation. Results are summarized by treatment groups which show the incidences seen in the original kidney section, the kidney step section review, and the combined incidence table of lesions seen in both the original kidney sections and in the step section review. Given the importance of renal lesions following gasoline exposure and the difficulties in evaluation, I would also recommend a Peer Working Group (PWG) on the kidney histopathology analysis. 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.

Discussion may wish to comment on the potential for early deaths or body weight

losses to affect the incidence of renal lesions in the high level BGVC exposed

rats. It is not clear what influence early deaths in the high dose males (mortality not significantly different from controls) or lower body weight (91.7% of control)

would have had on the

incidence of tumors in the high dose males. There was only a 2 percent difference in body weight between mid and high dose males, yet the highest incidences of

adenomas and carcinomas occurred in this group. No speculation regarding the influences of early mortality or lower body weight has

been added. No change

Page F-5, F-6: August column uses extra Appendix F is now Appendix G-5 & G-6

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significant figures for SD. G. SD entries on these pages

have been corrected.

Why are there differences in the number

of female rats examines in Table 3-12 (page 3-18) versus that noted in Table 3- 13 (page 3-19). The number of rats examined differed since the respiratory tract (a target organ) of all females in all groups was examined (Table 3-12).

Table 3-13 shows renal histopathology. Since the kidney was not considered a target organ in females (target organ in males only) all kidneys were examined in control and high dose groups, and only tissues with gross

lesions in the low- and middose groups were examined

at terminal sacrifice. No change.

211 (b) Research Group Comments 2

Page 2-11. Top of page. The first draft of the GMVC study contained an additional paragraph and Table (2-3) that discussed severity scoring of chronic nephropathy which were not included in this report. It seems that it should be since it was a finding in both

studies. The paragraph and table from the GMVC report is not being included in the BGVC report because the BGVC pathologist approached assessment of the severity of chronic nephropathy in a manner not totally consistent

with that indicated in the noted GMVC paragraph and

table. It would be inaccurate

to include these materials in the BGVC report as

indicative of the BGVC pathologist thinking process. The approach the BGVC pathologist used for assessing severity of CNP is located on page 4 of the pathologist’s
report. No change

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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Page 3-17. Histopathology – results are discussed as either neoplastic or degenerative changes whereas the GMVC report categorizes them as proliferative and nonproliferative changes. Please use consistent terminology between the two papers. Terms harmonized between reports to refer to changes as ‘proliferative and nonproliferative. 3-17, 3-22, Table 3-12, P-5, P-13

Page 3-18. Discussion of nasal epithelium degeneration. The BGVC report provides a comprehensive analysis of both the incidence and severity of lesions in the nasal passages

which is in contrast to the brief mention for the GMVC study. If the findings between the two studies are similar, they should get similar analysis and

discussion. There are also different conclusions drawn. The BGVC study indicates that the severity of the lesion in both sexes is likely induced by exposure to the test substance, whereas the GMVC study indicates that it is not attributable to MTBE, but rather the irritant properties of the fuel vapor

mixture. Results section and path report state that the nasal epithelial degeneration is likely due to the test article, but excludes identifying a particular property of the test article ( i.e. irritancy) as the cause. Reference to irritancy as a cause of nasal epithelial changes in the GMVC report

has been removed.

Are the two reports consistent in interpretation of similar findings for nasal respiratory epithelia?
Note – GMVC had increase, not decrease in olfactory epithelial degeneration, so only applies to respiratory epithelia findings

. 3-24 – 3-26; P-15 through P18

A protocol deviation for the change of statistical methods used to analyze histopathology results needs to be added. (It can be copied from the GMVC report.) Inserted. 4-4

Results and Conclusions 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. Results of pre-study chamber

trials are now included in Appendix E (Original Appendix D is now E). Appendix E-1

Pg 3-2: Homogeneity of Vapor

Concentration – raw data should be added to report as an appendix to substantiate the summary conclusions. Results of Homogeneity evaluations are currently in Appendix E. Appendix E-2

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Pg 3-15: Hematology

2 nd paragraph – delete the last phrase of

the last sentence since the differences at 18 months listed were not significant. Phrase deleted 3-15

Pg 3-16: Table 3-11 Hematology – “d” footnotes for values in the body of the table are probably mi slabeled and should be “e” (footnote d refers to trend analysis). Corrections made 3-16

Tables 3-5 and 3-6: Percent control body weights are incorrect for mid- and high doses starting at day 234 in both tables. Tables revised and checked 3-7; 3-8

Appendix N, Statistician’s report, pg N24 & N-25: histological findings and corresponding sta tistical analysis appears to be duplicative for data presented on pages N-22 & N-23. However, the data itself is different.

Which set of data is correct? Statistics tables have been totally replaced. Correct data are now included. Appendix N is now Appendix O. Appendix O

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). Individual animal gross findings at necropsy are now included in the pathology appendices for males and females, now Appendices M and N, respectively Appendices M-1 and N-1

211 (b) Research Group QA/QC

Reviewer

General Comments : Date of Site Visit and Review: June 8- 10, 2005

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. No response required.

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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.

The following items require further

consideration:

1. Page viii, Subcontractor: The protocol never indicated the use

of a subcontractor for the

statistical evaluation. This

should have been amended when

it was known that the statistical evaluation was going to be

contracted to someone other than the test facility. It should be written as a protocol deviation at this point. The address of the

subcontractor needs to be

indicated as well. Protocol deviation added to

text. 4-5, 4-6

2. Page x, OA Statement: Since a

subcontractor was used for the statistics, QA must assure the GLP practices and procedures of this subcontractor, so there should be a separate entry for the inspection/audit of the statistical report and data. QA audited statistics report, and separate entry has been added to the QA statement. xvi

3. 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 detailed descriptions of

audits have been added

. xiv - xvi

4. Page xi, and the Introduction, page 1-1: 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 Regulatory Guidelines/EPA regulations have been added.

LRRI has verified that of five serology assays performed by BioReliance, two were

conducted under GLP xvii, 4-3, Appendix C

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necessarily indicated in the protocol, that needed to be conducted in compliance with GLPs. Also it appears from the BioReliance Reports that not all Serology performed on Sentinel

animals was done under GLPs. Please verify. guidelines and three were

not. This has now been noted in the GLP compliance statement of the report and specified in the Appendix. Light and Sound

measurements were also not performed under GLP guidelines. This is also noted in the compliance statement.

5. 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 xvii

6. Page 2-1. Identification, Source and Storage: Original

characterization of the test substance was performed by

CRTC and ExxonMobil Biomedical Sciences, Inc. ExxonMobil Biomedical Sciences, Inc. has been

added. 2-1

7. Page 2-2, Animal Disease Screening Program: This paragraph is not complete with respect to all of the tests that were performed (also Lymphocytic Choriomeningitis Virus, Reovirus, CarBacillus, and parvovirus). The entire paragraph was replaced to be consistent with information provided in the GMVC report. Assays listed there were complete and more detail regarding sampling times were provided. 2-3; 2-4

9. 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 continuously but was not recorded, as stated in the report text. NA

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9. Page 2-3, Environmental

Conditions: The last sentence indicates that light and noise

levels in the chambers were determined periodically. Where are these data located? They should be reported. Light and noise levels are

now included with the

Environmental Data Results in Appendix G. Appendix G-1

10. Page 2-3, Diet and Drinking Water: Analyses of these parameters need to be kept with study data. These data are now provided in Appendix D (all subsequent appendices have been re lettered). Appendix D

11. Page 2-4, last line: should

indicate all Protocol-required

tissues. Text revised Table 2-2, page 2-6

12. Page 2-9, Organs and Representative Samples Taken for Examination: The Pharynx is listed here, but not found on tissues actually examined microscopically. It is assumed to

be reviewed with the section of larynx, but this should be

clarified. The assumption is correct, the pharynx was examined in conjunction with the larynx.

This is now clarified in the Tissue list provided in Table

2-2. Table 2-3, page 2-11

13. 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. Body weights indicated in the original text were both in life

and at terminal sacrificed.

Therefore the Path Tox statistical analyses were the

same for both circumstances.

This is clarified in the text on

the Statistics section. 2-13

14. Page 2-13, Histopathology: The protocol indicates that statistical

evaluation listed as number 2 (logistic regression) would beused, as well as the HoelWalburg test. The other tests (1, Cochran-Armitage and 3, Fisher’s exact) may be used as well, but the logistic regression A protocol deviation for

statistical methods had been

added – it is the same protocol deviation statement

listed in the second draft of the GMVC report. 4-4; 4-5

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and Hoel-Walburg test were protocol-required. Please

comment.

15. Page 3-1, Homogeneity of Vapor

Concentration : The second

sentence indicates that data obtained from an exposure day was compared to that obtained in

a reference location. The inhalation report indicates a

“reference location in the

chamber”. That makes it a little clearer. And the “exposure day” reference is confusing as this is during the pre-test trials. Please clarify. Text revision verified for accuracy. JB Nov 13, 2008 3-1

16. Page 3-2, Table 3-2, mid-dose

achieved concentration is

10.1 ± 0.37 (Table D-4 in Appendix D, page D-26; but is 0.34 in Appendix D, page D-11). Please verify the correct values. The correct value is 10.1 ±

0.34. Table 3-2, page 3-2, Table E-4, on page E71, and Section E-5, page E-56.

17. Page 3-3 and 4: The survival analysis is calculated omitting

the 8 females that were removed from the study for reasons not related to the BGVC exposure. So in Table 3-4, shouldn’t all 8

of these females be listed in the “other” category, but others should probably be listed there

as well. The table has been corrected.

Animals removed for nasal injuries or for trauma were

listed as moribund sacrifice animals as they were

euthanized. Table 3-4, page 3-5.

18. Page 3-4, Table 3-3: I cannot verify the mean days of survival figures reported in this table. Please verify that these values

are correct and/or data were correctly entered into the program that calculates this. The survival dates were

extracted from Path Tox and quality checked before being

sent to the Statistician. Some

animals were deleted from the analyses for various reasons, particularly early in the study. These are identified in the report text page 3-3 Table 3-4, and page 3-3

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Table 3-4 has been corrected to accurately reflect the animal disposition.

19. Page 3-6, Body Weight Gain: Body weights of mid-level females were significantly below control values from Day 122 to the end of the study (except day 318 and 378)..... Text revised 3-6

20. Page 3-7: Indicate what

statistical test was done here in

the footnote. Statistical test covering body weights have been added to

the footnotes of Tables 3-5

and 3-6. 3-7, 3-8

21. Page 3-11, Table 3-8: The

footnote indicates statistical significance at the p ≤ 0.05, but the female kidney in the high dose and the male epididymis in the mid dose are significant at the p ≤ 0.01 level. The same is true for Table 3-9 on page 3-13 for the female brain in group 4, the female heart in groups 3 and 4, the female kidney in groups 3 and 4, the female liver in group 4, the female lung in group 4, and the female spl een in group 4. Also, Table 3-10, page 3-14 for the female kidney for group 4. It also appears that the female lung in group 4 is not significant. Please verify. No change – protocol specified a significance level
at p< 0.05.

Study Director has cross

checked with the Path Tox Printouts for female high dose lung to brain weight

ratio and it is not different

from control. The table was revised accordingly. JB

12/23/08. 3-14

22. Table 3-11, Summary of Hematological Findings:

Shouldn’t the footnote “e” be added to the 18 month finding in

females of Absolute Band Neutrophils, as the P value is 0.04 (noted in the statistical report, Table N-2b). Table Corrected. 3-16

23. Table 3-11, Summary of Table Corrected 3-16

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Hematological Findings:

Shouldn’t the footnote “e” be added to the 12 month finding in

males and females of Nucleated

Red Blood cell as the P value is 0.002 and 0.03, respectively (noted in the statistical report Table N2a and N3a).

24. Page 3-15, Hematology, second paragraph: The values reported by Charles River in the

referenced website are means

and not medians. These values may not be comparable. True; reference to Charles

River website deleted. 3-15

25. Page 3-15, second paragraph: The differences resulting from blood sampling technique should be further explained. Different groups were sampled similarly, correct? Was it just that the technique itself may have been faulty or variable? If this is the case one may not be able to draw any conclusions from the data. Text has been modified with concluding statement that the significance of the changes at

12 months is unknown. 3-15

26. Page 3-15, third paragraph, first

sentence: This sentence should indicate that the high-level male group at the final sac...... The next sentence is missing the upper range of the high dose level (it is 189.6 x 10 3 cells....) Added reference to males and upper value of cell number

range. 3-17

27. Page 3-11, 24 Month interval, Males, Nucleated RBC: The range for the control group should be (0.0,21.0). Corrected in Table 3 - 11 3-16

28. Page 3-11, 12 Month interval, Females, Nucleated RGC: The range for the high dose group should be (0,5) not (0.5).. Value of 0.5 has been entered

for Table 3-11. 3-16

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29. Page 3-18, first sentence: degeneration of the nasal epithelium.....Is present at minimal-to-mild severity in all groups. Moderate severity was only toted in the group 3 and 4animals. (Appendix O, Page O10, first sentence also). No change; data in table makes this point. Appendix

O is now P. 3-24; P-15

30. Page 3-18, fifth sentence: It is

not clear what this sentence is referring to Table 3-13, and there is no other significance noted for the male turbinate level 2. (See also Appendix O, Page O-10, first paragraph). No change; paragraph is discussing male data and this is the concluding remark with respect to nasal epithelial degeneration at level 3. 3-24; P-15

31. Page 3-20. second paragraph, first sentence: BGVC exposure caused a statistically significant exposure-related decrease in the incidence of degeneration....(see also Appendix O, Page O-10, last paragraph). No comment to address 3-24; P-17

32. Page 3-20. second paragraph,

second and third sentence: The incidence and severity were both decreased male and female high doses, and at mid dose in females. (see also Appendix O, Page O-10, last paragraph). No change; text clearly states results correctly. 3-24; P-17

33. Page 3-21, Conclusions: The first sentence should identify the

test material as Baseline Gasoline Vapor Condensate as it has been referred to throughout the report. Text corrected 3-27

34. Page 4-3, Gas Chromatographic Analysis: The potential impact

of the deviation mentioned in the second paragraph is not given.

The same is true for the last

sentence in the collection of Text added to indicate deviation was judged to have no significant impact on study 4-3, 4-4

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Organ Weights at Necropsy.

34. Appendix N, Table N-14, page N-20: For the tissue Ovaries, Group 4, how could the N be 50 for atrophy? Only 49 were examined for other diagnoses. N = 50 for ovaries. The statistician was provided with

corrected tissue data and the statistics were reanalyzed. Appendix N (now O)

contains the corrected tables. O-20

35. Appendix N, Table N-5, page N29: For group 4, Inguinal Lymph Node, Sinus dilation, how could the N be O, when this

tissue was reviewed in one

animal? Shouldn’t the incidence

be 0/1? The statistician was provided

with corrected tissue data and the statistics were reanalyzed. Appendix N (now O)

contains the corrected tables. O-27

36. Appendix N, Table N-5, page N30: For Group 3 Larynx, how

could inflammation incidence be 31/50 but the neoplastic

incidences are 1 and 0/49. The statistician was provided

with corrected tissue data and the statistics were reanalyzed. Appendix N (now O)

contains the corrected tables.

Denominator is 50 for all. O-28

37. Appendix N, Table N-5, page N32: For the tissue, Lymph Node, other, group 2, there are

incidences with Ns of 6 or 7. Why are they not all the same? I believe they should all be 7. The statistician was provided

with corrected tissue data and the statistics were reanalyzed. Appendix N (now O)

contains the corrected tables.

All denominators are 7. O-30

38. Appendix N, table N-5, page N38: For the tissue, prostate, control animals, there are

incidences with Ns of 49 and 50.

I believe these should all be 50. The n for control group prostate is 50. the table N-5 (now O-5) has been

corrected. O-36

39. Referencing No. 27-31 above: It

is unclear how these discrepancies could have

occurred. If these tables were computer generated, the N values could only have been the number that were examined, and

could not be different with the

same tissue. Please comment. The source of the errors in

numbers of animals in certain groups (see No 27 – 31), was that the pathologist did not “flag” these tissues in certain animals as being read. These errors were not caught during QC review before the

data were extracted and sent NA

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to the statistician for analysis.

40. If these (above referenced tables) were not computer generated but were word processed, additional auditing needs to be performed

to assure that all data are

accurate. The statistician has re run the analyses and the corrected

tables have been checked to verify the correct animal numbers (n’s) are present. QA has audited the statistician’s report. xvi

41. Appendix N, Table N-5, page N39: Under the skin tissue entry, why re mammary, preputial and Zymbal’s gland neoplasms

listed? Shouldn’t these be

summarized with their respective

tissues? The mammary, preputial and Zymbal’s gland lesions were originally identified as skin lesions at necropsy. They

were maintained in that
category so gross-tomicroscopic correlations could be made. Rearranging

the results to include these findings with their respective

tissues would not alter

statistical outcomes or the

bottom line. No correction proposed. No change; O-37

42. Appendix O, page O-4: The 13 Week Subchronic Study with

BGVC should be footnoted. Is

this the one Lovelace started and

aborted or the one HLS performed? Footnote has been added to appendix O, now Appendix

P. P-13

43. Appendix O, page O-13: One diagnosis that appears to be statistically significant, yet not discussed, is the mastocytosis of the Mesenteric Lymph node in

the males. Please comment. No change; pathologist dismissed finding as “. . . the biological effect of the BGVC exposure on these incidences is not apparent.” P-11; P-12

44. Appendix O, Page O-14, last sentence of first paragraph:

There was a statistical difference between the mid and high-dose group, so this statement should

be amended. No change; there were no significant differences when using the Bonferroni correction for pairwise comparisons. Appendix P, No change

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45. If one compares the last clinical observation to the necropsy observations, it appears that the

last clinical observation of “skin growth” frequently was neither

confirmed nor denied at necropsy. One can only assume

that the skin that the skin growths noted clinically were in fact preputial masses or enlargements. This should be

mentioned in the text of the report, as it may seem that these “skin growths” have been lost.

Please comment. In life clinical observations

recorded in Path Tox did not necessarily follow the same format as recording gross lesions at necropsy on IANR forms. In the former case, examples of Path/Tox

notation of skin lesions

include “Hair/skin/skin 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.

46. Gross to Microscopic Data

Table: Animal number 226 has a gross observation of Lung, right

cranial lobe? (cardiac) The data in the IANR form

for animal 226 were verified by the study pathologist. The gross observation was indeed for the right cranial lobe. No

correction needed. No change

47. Gross to Microscopic Data

Table: Animal number 261 (group 3 female), the gross observation of “pituitary gland discoloration, 0-2 mm, dark focus, irregular” was not on the necropsy sheet. Could this have

come from another animal? This appears to be a data entry error. It has been

corrected. N-341

48. Appendix J, Organ Weights: The uterus weight for animal number 372 should be 0.872g, not 0.372. Summary tables will This appears to be a data entry error. It has been

corrected in Path Tox and revised reports are included K-15, K-30, K-44, Tables 3-8, 3-9 and 3-

10.

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also be needed to be corrected. in the appropriate Appendix.

49. Appendix J, Organ Weights: The liver weight for animal

number 287 is over twice that of others in the group. Gross observations do not support sucha weight. It should have been reweighed. Means and statistics,

calculated both with and without this value may be useful. K287 was a final sacrifice

female. Gross lesions were observed “discolored, patchy, mottled (3+), irregular and pale”, although no specific notation of “enlarged” was made. Histology indicated biliary epithelial hyperplasia, fibrosis, mononuclear leukemia (with mononuclear cells replacing much of the parenchyma), and hepatocellular necrosis. Liver weights were highly

variable in this mid-level group with weights ranging from 7.537 to 20 .848 g (K287). The next highest liver weight was 14 grams. Of note, the kidneys, ovaries and spleen for this animal were also noted as being “high” by Path Tox. The spleen of this animal weighed 10 grams, The spleens of 16/27 females in the group were less than 1 g. Animals having higher weight livers also had higher weight spleens (over one gram). For example, female K261 whose liver weighed 14.766 g had a spleen weighing 14.669 g. Therefore, omission or rerunning the statistics on

animal K287 does not seem appropriate. The high liver weight is consistent with a high spleen weight and likely secondary to leukemia. No change

50. Pathology Raw Data Listing (Appendices L and M): The Symbols and Abbreviations list

does not include some of the

notations contained in the Raw Data Listing. For example, Definitions of Codes are now included in the Pathology Appendices (M and N). M-364, N-314

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under Death Code, there appears U1 and U2. What designation is that? As well, there is a small

case s and p next to many of the diagnoses. What does not

denote?

51. Pathology Raw Data Listing:

The use of “U” should denote

tissues that were not histologically remarkable. One

would think that this means anything that was within normal ranges would have a “U” entered, but his is not the case. Sometimes it is entered, seemingly only for the esophagus and sciatic nerve. Since this is the only way to verify that the tissue was examined, why is it not entered

for all tissues that do not have a finding? Presently one is not

certain that tissue was examined if there is no diagnosis. In cases where a tissue (sciatic nerve or esophagus) has no finding (lesion) in any animal of a given sex and exposure group, the symbol

U is indicated for that tissue.

U indicates a tissue was examined and had no finding in any animals of a given sex and exposure group. If a

lesion is noted in one or more animals of a given sex and group, that finding is listed under the organ heading and

the lesion is scored or

otherwise noted for those

animals For animals in the same group not having that finding, a dash is presented to

indicate that he tissues was examined, but no lesion was
present. No change

Additional Raw Data/Report

Comments

1. Gross to Microscopic Correlation,

Female No. 258: The trim sheet indicates that the mammary mass is missing. The Gross to Microscopic

Table describes the mass and the microscopic comment indicates “no correlation entry made”. The Pathologist’s comment sheet indicates

that the trim record indicates the mass was lost. Please clarify as it appears that the Pathologist reviewed the mass and had no correlation diagnosis. In this case, the mammary

mass was lost between necropsy and trimming. The

Path Tox Gross to Microscopic Table notation

that “no correlation was made” simply means that it is impossible to correlate the gross finding to a histological finding because there was no sample to analyze. The pathologist was aware the sample was lost. No change

2. Trimming records for animals 69 In this case the pathologist No change

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and 317 indicate that one adrenal was missing. There is no mention of this for #317 on the Pathologist’s comment sheet or the Gross to Microscopic correlation table. For animal 69, the Pathologist indicates that a recut was examined. Please clarify. was inconsistent in noting of one of a pair of tissues (as adrenal) was missing. Since the pathologist was able to

read one of the two adrenals (in the case of animal 69, the

block was recut to obtain a sample to read), Data were

obtained. A recut is performed on a block when an adequate tissue section is not obtained. A recut will go deeper into the paraffin block and provide another opportunity to obtain a useful

section to stain and read.

3. The trimming record for number 52 indicates that the urinary bladder is missing. This is not indicated on the Pathologist’s comment sheet or on the Gross to Microscopic Correlation Table. Please clarify. The bladder is noted as missing in the Path Tox missing tissue report. Since it was not noted as missing in the Pathologist’s comments, no Gross to Histo report comment was generated.

4. The trimming r ecord for animal number 93 indicates that nasal passages are missing. This is not contained on the Pathologist’s comment sheet or on the Gross to Microscopic Correlation Table. Please clarify. As per the pathologist, nasal tissue and pituitary were not present at trim and no block or slide was prepared. Path/Tox entry indicates the tissues were missing. A

deviation has been added to the report covering these

omissions. 4-6

5. The trimming r ecord for animal

number 134 indicates that there is no optical nerve with the eyeball. This is no comment on the Pathologist’s

comment sheet or on the Gross to Microscopic Correlation Table. Please clarify. By convention at LRRI, it is agreed that having at least one optic nerve to view microscopically on sections from one pair of eyes (or one

adrenal medulla out of two adrenal glands, one parathyroid gland embedded in a pair of thyroid glands, one tracheobronchial lymph node out of 2 or 3 that may be present in the segment of

caudal trachea/tracheal No change

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bifurcation) is sufficient, because histosectioning is too

difficult to achieve both.

When this has been the case

not all of these were entered into PathTox as “one of pair missing”.

6. Animal number 320, Day 591, “enlarged spleen” was to be added 3/31/0303, but was not done The animal didn’t die until day 605, so it is unclear why it was to be added. The note to add

this comment should be crossed out. We were unable to locate any

note reference the need to

add a comment that the animal had an enlarged spleen. Possible the

comment was identified earlier as an error (or a notation for another animal). The spleen weight on this animal was 1.188g, terminal body weight 419.4 grams. Spleen weight is not consistent with it being enlarged. No change

7. Animal 309, Day 591, “Added facial injury” does not appear in the
report. The facial injury was secondary to the presence of

a tumor in the animal’s head

that is documented in the histopathology findings for

this animal. No change

8. Clinical Observation Table for

animal number 120 indicates None/Dead for Days 438 and 451. Shouldn’t death only be recorded once? As well, it appears that data may have been

recorded after this animal died. Please clarify. The animal had died on day 437, but because the animal’s

death was not recorded in path tox immediately (i.e., removed), his number came up for clinical observations on days 438 and day 451. Therefore, the only option

available was for the staff to enter “no observations,

animal dead”. No change

9. Clinical Observations: Day 626,

Animal numbers 151 and 152: Records

indicate that observations for that interval were deleted, but they are still in the report (eye, opacity and The observations have now

been corrected in Path Tox and a new, revised report included in Appendix J. J-32

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microphthalmus and jaundiced skin).

10. Clinical Observation: Animal number 72, Day 318, week 46: This

animal is dead and should not have a body weight. Please verify. There is no body weight for I 072 for day 318 or any subsequent day. I-28

11. Clinical Observation Report, Animal 179. This report doesn’t include FD on Day 617. Last clinical observation was on Day 612, shouldn’t

it include FD? The animal was removed from the system before Day 617, so weigh crew was not prompted to observe on day

617/ No change

12. Animal number 341, Clinical observations, Day 542: “movements/activity/circling and jaundiced” were to be deleted but they were not deleted from the report. “Emaciated and hypoactive” were to be added but they were not. The error has now been corrected, a new Path Tox report generated and included in the revised Appendix J. J-24

13. Hematology Data, Female Number 371, 12 Month interval: It is

indicated in the data for Number 371 – “Unable to report data”. The report just skips this animal. This animal needs to have some explanation for not having any hematology data reported at the 12 month interval. As well, for animal 372,

there is no WBC estimate with no explanation. It is indicated for other animals that the slide quality was poor.

Is this the case? All hematology data have

been reviewed for completeness. A notation is

made in the cover sheets for each time point indicating

animals for which no blood

smear was obtained. Data for groups I and J at the 24 month time point were not originally included in the report, but have been added.

A protocol deviation has been

added to indicate lack of

blood smears on animals on study. Appendix L, L-2, L-15, L-28, L-41, 4-6

14. Necropsy sheets for the sentinel animals were completed inconsistently.

Sometimes there are no tissues checked off, with a notation of “no necropsy per protocol” and others have some tissues

checked and still others have all tissues

checked off as normal as a result of a full necropsy. Since the protocol is Amendment 2 (August 1, 2003) indicated that sentinel

animals were not to receive a complete necropsy at

terminal sacrifice. The protocol did indicate that any animals that died early were to undergo a full necropsy. No change

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unclear on what was to happen to these animals when they died or were sacrificed moribund, it is hard to say

what should have been done. Amendment 2 did not specify that sentinel animals dying or

euthanized before terminal

sacrifice were not to receive a complete necropsy.

15. Test Material Tank No. BG09: It appears that this tank was filled on 7/1/03 with a final weight of 19,412 g, but then was filled again on 7/18. It was the used on 7/22 beginning with a weight of 19410 g. Please clarify. Tanks were generally used only for one exposure day

and the refilled. The SOP on tank filling indicates that the tank was to be vented just prior to filling. Therefore the final weight recorded at the end of an exposure day will generally be greater than the immediate pre-fill weight.

The difference is the amount of vapor released by venting.

This amount was variable

. No change

16. Test Material Tank No. BG08:

This tank was used on 7/15/03 with a final weight of 13,703 g. The initial tank weight for filling was 13,345 g? Please clarify. See response for 15, above. No change

17. Test Material Tank No. BG07 was used on 7/9/03 and had 13,850 g left. At its fill on 7/22 it weighed 13,030. Please clarify. See response for 15, above. No change

18. Test Material Tank No. BG11: The initial and final weights are both 19510 g on 4/30/03 even though 5,505 g was used? (should be 14,005). Also on 7/22/03 the weights between last use and

fill no not correlate. This was a data entry error

and has been corrected in the

raw data. NA

19. Please also check the following tanks on the following days: Tank BG12 – on 7/11 (last use vs. refill weights) Tank BG17 – on 7/14 (last use vs. refill weight on 7/22) See response to 15, above. NA