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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 what 

would 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 report and pathologist report in documents with suggested revisions. Once LRRI revises the 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 typically 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 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. 

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 is 

discussed 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 lack 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 Aerosol 

Confirmation 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 as 

compared 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 counts 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 the 

quality of the protocolrequired tissues, the nonrequired tissue was not 

removed during trimming. If 

trimmed 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 certainty 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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RG responses to EPA comments 

Comments Action/Response Page #1

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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Comments Action/Response Page #1

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 α2-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 Comments2 

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 the 9/2/04 draft or were duplicative of EPA/independent peer reviewer 

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 

2nd 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 mislabeled 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 statistical 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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Comments Action/Response Page #1

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 be 

used, 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 spleen 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 103

 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 4 

animals. (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 such 

a 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 record 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 record 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