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“Baseline Gasoline plus Ethyl Tertiary Butyl Ether Vapor Condensate: A 13-week Whole-body Inhalation 

Toxicity Study in Rats with Neurotoxicity Assessments and 4-week In Vivo Genotoxicity and Immunotoxicity 

Assessments (by Huntingdon Life Sciences, Study No. 00-6129m Sponsor No. 211-ETBE-S).

EPA 

ORD finds that in general, the main study and the 

specific neurotoxicity, genotoxicity, and 

immunotoxicity assessments included in the 

report were conducted according to the EPA test 

guidelines. At this time, however, we cannot 

recommend approval of the study report as is, 

because we have identified significant 

deficiencies in the statistical analyses and 

reporting of data in the functional observational 

battery (FOB) and motor activity assessments. 

Similar issues were described previously in our 

memorandum to you of June 20, 2002, regarding 

the subchronic toxicity study for baseline 

gasoline. In addition, we are recommending that 

more data be collected for the micronucleus 

assay. The attached memorandum provides 

specific comments on the study report. 

This was the original draft report of these 

BG+EtBE VC studies and did not include the 

revised statistics for sister chromatid exchange and 

neurobehavioral endpoints recommended by EPA 

after reviewing the Baseline Gasoline Vapor 

Condensate submission. The revised statistical 

evaluations have been completed and are included 

in the second draft report of this study.

Additional data were collected for the 

micronucleus study and findings are described 

with the study review. 

Our review was conducted by the ORD 211(b) 

Health Effects Team, comprised of scientists 

from our National Health and Environmental 

Effects Research Laboratory (NHEERL) with 

assistance from the National Center for 

Environmental Assessment (NCEA) and the 

Office of Science Policy (OSP). 

ORD reserves judgment on the interpretation of 

the results, and conclusions drawn from them, 

until a formal assessment of the 211(b) findings 

and any other pertinent information is completed 

at a future date. We strongly encourage the RG 

to address our comments before final versions of 

the reports are submitted to EPA. The RG should 

also adequately respond to the comments raised 

by the independent peer reviewers and provide 

the additional information requested.

No comment needed

At your request, NHEERL scientists have 

reviewed the submissions from the American 

Background information. No comments needed
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Petroleum Institute (API) entitled, “Gasoline 

ETBE Vapor Condensate: A 13-week Wholebody Inhalation Toxicity Study in Rats with 

Neurotoxicity Assessments and 4-Week In Vivo 

Genotoxicity and Immunotoxicity Assessments.” 

The report was submitted in three volumes from 

Huntingdon Life Sciences (Study No. 00-6129, 

Sponsor No. 211-ETBE-S) with additional 

reports from other subcontractors to API, 

including reports on neurotoxicity assessment 

using GFAP, immunotoxicity, and genotoxicity 

assessment using sister chromatid exchange and 

micronumcleus assays. In addition, NHEERL 

reviewed the reports of two peer reviewers of the 

studies and the API response to the peer review 

comments.

While in general the NHEERL reviewers 

concluded that the studies were conducted in 

accordance with the testing guidelines, there 

were also significant exceptions involving the 

conduct of some of the studies and analysis of the 

data. These issues were described previously in 

our memorandum to you of May 21, 2002, 

regarding the baseline gasoline study. We 

understand that these continuing issues will be 

addressed by API in a revised final report. In 

addition to the issues previously noted, we have 

the following assessment of the gasoline plus 

ETBE study reports.

Sister Chromatid Exchange Assay and 

Micronucleus Assay

The micronucleus assay appears to be properly 

conducted according to the required guidelines. 

The conclusion in the report that “G+ETBE VC 

may cause an increased incidence in 

micronucleated polychromatic erythrocytes” is 

supported by the results. However, given the fact 

that the significant outcome was somewhat 

dependent on the statistical analysis method used, 

it is appropriate to collect data from a second set 

of slides. This is being done, and it is hoped that 

the addition of the data acquired will make the 

interpretation firmer.

A second set of slides from the micronucleus assay 

was read and the entire report revised to reflect the 

additional data. Although there was an apparent 

increase in group mean micronucleated immature 

erythrocytes (mie) with increasing concentrations 

in the second set of slides, none of the values from 

animals exposed to BG+ETBE Vapor condensate 

were statistically significant compared to negative 

controls. The report has been re-written as draft 2 

to include these data.
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The sister chromatid exchange (SCE) assay 

appears to be conducted according to the required 

guidelines and the conclusion that BG+ETBE VC 

did not induce SCE is supported by the data. 

There is one proviso – as with other studies in the 

BG set – namely, that additional statistical 

analysis (1-tailed Dunnett test) be performed, 

since there was some concern with reliance on a 

trend test given the nature of the data. These 

additional statistics spear to be in progress.

Additional statistical analysis was performed and 

included in draft 2 of the report.

Immunotoxicity

We concur with the review by Drs. Schlesinger, 

Goldsworthy, Twerdok, as well as with the 

results obtained by Dr. K. White, who performed 

the immunotoxicity evaluations. The data 

indicate that the exposure to Gas+ETBE vapor at 

10,000 and 20,000 mg/m3suppresses the 

antibody-forming cell response, compared to 

vehicle control. This suppression of the antibody 

response to sheep erythrocytes occurred in the 

absence of any decrease in body, spleen or 

thymus weight. As such, the data indicate that 

the immune system in rats, exposed at the two 

highest doses, is unrelated to a generalized overt 

toxicity and suggests that GAS-ETBE may target 

the immune system.

A minor point is that Dr. Goldsworthy, in his 

review of the “Immunotoxicology Evaluation...,” 

stated that “Gasoline+ETBE vapor condensate 

did result in a dose-related decrease in the spleen 

IgM antibody-forming cell response to sheep 

erythrocytes.” In fact, there was no “doserelated” decrease, since the IgM AFC/106

and the 

IgM AFC/spleen data demonstrated that there 

was a slightly more pronounced suppression in 

the AFC at 10,000 mg/m3versus that at 20,000 

mg/m

3

. This is observed in the data presented in 

Fig. 4 and Table 2.

These findings were reported to EPA under 

Section 8(e) in 2002.

The EPA reviewer is correct. No changes were 

made in the report.

FOB and Motor Activity

This protocol has been reviewed previously with 
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the studies for baseline gasoline, and gasoline 

containing MTBE, ethanol and Tame, and the 

same issues apply here. After the first baseline 

gasoline study, we discussed those issues with 

the sponsor and the contractor, and they are being 

addressed. However, since these studies were 

actually conducted prior to those discussions, the 

changes have not been made in the current 

document. Therefore, the outstanding issues are 

only summarized here.

Revised statistics for neurobehavioral endpoints 

were performed and Draft 2 of this report rewritten 

accordingly.

Overall, the study was conducted in compliance 

with the published EPA test guidelines, with the 

exception that some endpoints (e.g., reactivity 

measures, gait score, sensorimotor responses) are 

not ranked, as is required. The statistical analysis 

of the neurobehavioral data is not acceptable in 

its present form, but the sponsor’s statistician is 

working on more appropriate analyses. 

Specifically, both motor activity and FOB data 

should include repeated measures, and grip 

strength and foot splay duplicates should be 

averaged and the analyzed. Sex should be 

included as a factor in the overall ANOVAs for 

all endpoints. 

See comment above

A review of the actual data revealed no obvious 

differences across treatment groups. There were 

a few differences pretest, but these were small in 

magnitude and were not evident with subsequent 

testing. Provided that appropriate statistical 

analyses do not raise new questions, we accept 

the results of the study.

Application of revised statistics did not alter the 

conclusions of these tests. No neurobehavioral 

effects were observed.

Neuropathology

We have examined the neuropathology sections 

of the study, and based on the information 

provided, we conclude (a) the neuropathologic 

assessment was conducted in accordance with the 

EPA Neurotoxicity Screening Battery Guidelines 

and (b) the data are consistent with the 

conclusion of the report that there is no evidence 

of treatment-related neuropathology in the 

No comment needed
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20,000mg/m3exposure group. The reviews by 

Drs. Goldsworthy and Schlesinger are consistent 

with our evaluation.

General Conclusions

In general, it appears that the studies were 

conducted in accordance with the appropriate 

testing guidelines. The study reports were 

prepared before API received EPA comments on 

the reports from the baseline gasoline vapor 

condensate 13-week study, and so a number of 

issues raised in our review of that study recur in 

the current report. We understand that those 

issues will be addressed in a revised report, in 

action to the issues raised here specific to the 

gasoline plus ETBE 13-week study. Overall, we 

agree with the study, the peer reviewer comments 

and API’s response to those comments. Among 

the issues that still need to be addressed for the 

gas plus ETBE study are the analysis of second 

set of slides in the micronucleus assay and 

additional statistical analysis for the SCE assay, 

both of which appear to be planned or are 

currently being done.

See comments above.

Peer Reviewer – Schlesinger

Overall, the main study was conducted in a 

scientifically sound manner and followed the 

appropriate protocols. There were no significant 

deviations from these protocols that would have 

affected the outcome of the study. However, a 

number of concerns related to statistical analysis 

and the basis for interpretation of toxicological 

significance are indicated.

The conclusions are generally sound, although in 

one specific case there could be some misinterpretation of results. This involves the conclusion 

in the main study section of the report that the 

10,000 mg/m3level is considered to be the 

NOAEL. However, there seem to be some 

effects on some parameters at this exposure level 

from results reported in the main study, and there 

clearly is an effect at this level in the plaque 

The statistically significant clinical chemistry 

changes at 2000mg/m3cited by Dr. Schlesinger 

tended to occur in one sex only, a treatment related 

pattern was not apparent and absolute differences 

were small with no corroborating histopathology. 

The Immunotoxicity study was performed with 

animals exposed for 2–weeks during the 13-week 

study and data were analyzed separately, 

demonstrating significant effects at 10,000 and 
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forming immunological assay. An individual 

reading just the main summary may, thus, arrive 

at a false conclusion as to the biologically 

effective level of the vapor.

As noted previously, it would have been 

preferable if all of the satellite studies employed, 

to the extent possible, comparable statistical 

approaches to data analysis. They basically all 

had the same study designs and could have used 

similar statistical assays. This would have 

avoided the potential for different statistical 

techniques to have different sensitivities.

The conclusions reached in the micronucleus 

study are generally supported by the data 

presented.

The conclusions reached in the GFAP study are 

supported by the data although the authors seem 

to imply that the results are quite nondefinitive.

The conclusions reached in the SCE study are 

supported by the data.

The conclusions reached in the immunological 

assay are supported by the data.

Detailed comments for each of the draft reports 

are attached.

20,000mg/m3. The “main study” conclusions 

reflect only the results and NOAEL of the 

subchronic toxicity study. A table has been added 

to Draft 2 summarizing the results of all satellite 

studies so that the reader will be fully apprised of 

NOAELs for different endpoints. All satellite 

studies are supplied as Appendices to this report.

While recognizing the value of this comment, the 

RG must employ different contract laboratories 

with recognized special expertise to perform 

satellite studies with different endpoints. Each 

laboratory employs an individual statistical 

package developed by experience and the 

historical database for the assay. Since most of 

these packages are computer-based, it would be 

resource intensive and costly to require the 

subcontractors to employ statistical methods that 

are not standard practice in their laboratory. In 

addition, comparison of current data to historical 

databases within a laboratory, where appropriate, 

would be more difficult if statistics were not 

consistent.

MAIN STUDY

Section 2.20.1 Method of Analysis (p. 43)

When the Bartlett’s test indicated nonhomogeneity of variance the investigators used a 

A single computerized statistical testing package 

was employed for all parameters listed on p.43-44 

of report. In this package, analyses by individual 
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nonparametric analysis of variance. An 

alternative approach would be to use some 

transformation that would have resulted in 

variance homogeneity, and then a parametric 

analysis of variance could have been used. This 

would have made the statistical analysis of all 

data sets much more internally consistent and 

would not present any potential for different 

types of tests having different degrees of 

conservativeness in detecting any significant 

changes from air control. In any case, the 

investigators should indicate which specific 

statistical tests were used for which datasets.

sex or sexes combined were programmed to be 

performed separately by one-way ANOVA, rather 

than analyzed together by two-way ANOVA. 

Terminal and recovery data were also analyzed 

separately. After discussion with the study 

director, it was decided that additional justification

for the statistical method employed routinely for 

repeat-dose studies throughout this testing facility 

was probably not needed.

The use of the one-way ANOVA as described 

infers that when there was more than one time 

point for analysis, e.g., terminal and recovery, 

that data for each endpoint was analyzed 

separately at each of these times. It would have 

been possible to use one evaluation that could 

have included the various time points and both 

genders in each analysis for each endpoint. A 

clearer justification for the statistical methods 

actually used by the investigators should be 

provided.

See comment above.

What was the rationale for performing statistical 

tests at two levels of significance. In the satellite 

studies, for the most part only one level was 

used. This difference could lead to 

interpretational problems.

Contract laboratories provide both levels of 

significance to satisfy needs of a range of clients 

and both values are often identified in standardized 

statistical packages for computers.

Section 3.1. Chamber Monitoring. (p. 47):

The table on page 48 shows that the air control 

chambers as well as the test chambers contained 

particles. What were these and why were there 

not appropriate filters on the chamber intake to 

remove particles from the incoming air?

The mean particle distribution was comparable 

between controls and treatment groups. 

Appropriate filters were in place but some particles 

are invariably present in chamber atmospheres, 

representing background air. 

While the data support the conclusion that the 

gasoline atmospheres were in vapor form and not 

in particulate form, has any thought been given to 

the possibility that the small “background” 

particles which were also present in the vapor 

It is unlikely that test vapor would condense on 

these particles, given the volatility of these light 

end vapor components, and the high level of 

consistency in constituents proportions 

demonstrated analytically from week to week. In 
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atmospheres could have acted as modalities for 

the condensation of the of the vapor and a vector 

to areas it may not have reached if inhaled in the 

absence of such particles?

addition, no unique particle disposition was 

observed microscopically in lungs or nasal 

passages.

Furthermore, from Appendix A it appears that the 

particles have a wide size range, from <1 to >9 

micrometers. Finally, it is stated on page 48 that 

the results “ indicated that the atmospheres were 

essentially vapor only...since there was no 

substantial difference between the test substance 

chambers and the air control chambers for 

particle content. This statement does not seem to 

make sense. What the data show is that all 

chambers had some particles.

It is agreed that the data showed that all chambers 

had some particles. The sentence in question 

should convey that the absence of higher 

particulate mass in weekly air samples from test 

substance chambers compared to control chambers 

supported the likelihood that introduction of 

BG+ETBE vapor condensate did not contribute 

additional particulate material into the chamber 

atmospheres, and thus could be considered all 

vapor.

On page 47, mention is made of the possibility of 

some inaccuracy in the calibration of the IR 

monitor. This would be an unacceptable source 

of experimental error, since exposure atmosphere 

monitors should be accurately calibrated prior to 

the start of any study.

Average nominal concentration varied slightly 

above analytic concentration (low and mid doses; 

2,000mg/m3and 10,000mg/m3), to slightly below 

analytic concentration (high dose; 20,000mg/m3). 

Nominal concentrations are calculated by 

measuring the weight differences in the 5 gal. 

cylinder from which the test sample is dispensed, 

divided by the total vol. of air (m3) passing through 

the chamber. Large differences between nominal 

and analytical concentrations are useful in 

identifying possible leakage in sample delivery or 

chamber distribution, and could suggest 

disposition of test material on chamber walls, 

reducing the amount of material in the breathing 

atmosphere. Where variations are small, 

significance is questionable. In this study, 

differences are small and the analytical 

concentrations very closely match the target 

concentrations, providing a reliable determination 

of exposure. Calculation of nominal concentration 

can be most affected by the accuracy of calibration 

of the airflow rate, making calculation of nominal 

concentrations a less accurate measure of chamber 

concentrations than analytical determinations. 

The calibration of the IR monitor is the more 

accurate calibration than the chamber airflow 

calibration because the IR monitor calibration 

involves the precise injection (using gastight 
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syringes) of fixed volumes of test substance into a 

known and fixed volume of air. By comparison, 

the chamber airflow calibration involves the 

measurement of large volumes of air (~200 LPM) 

using a mass flow meter, a procedure that is 

inherently less accurate.

Summary and R/D clarified that nominal and IR 

results were in ‘acceptable’ agreement.

Section 3.8.1. Hematology (p. 50)

It is stated that exposure related differences seen 

at week 4 are no longer apparent at the terminal 

sacrifice interval. However, examination of the 

data suggests that there still is some decrease in 

reticulocyte count in females at the terminal 

interval sacrifice.

The decrease in reticulocyte counts seen in high 

dose females at terminal sacrifice was not 

statistically different from controls probably due to 

the large standard deviation and thus was not 

considered to be significant. According to the 

study director, the absolute differences in 

reticulocyte counts for high dose females 

compared to controls decreased from 18% at wk 4 

to 9% at wk 13, resulting in a decrease in statistical 

significance.

Section 3.8.3. Clinical Chemistry (p. 51)

A statement in this section seems to imply that if 

statistically significant changes occurred in only 

one gender then they would be considered to be 

toxicologically insignificant. This precludes the 

potential for gender-specific real effects. Also, 

some of the significant differences in these 

parameters seem to result from wide variability 

in the standard deviations of values rather than 

from any differences in mean values.

The statement on p. 51 of the report concerning 

discounting the effects seen in only one sex was 

not meant to diminish the potential significance of 

gender-specific effects in general but only to 

indicate that, in instances of random clinical 

chemistry changes, isolated occurrences in only 

one sex was one parameter for rejecting statistical 

significance alone as biologically important, 

especially if a treatment related pattern was not 

apparent, absolute differences were small with 

wide standard deviations and there was no 

corroborating pathology. 

Section 3.9 Organ Weights (p. 51)

Organ weight data presented in Table 12 appear 

to have been analyzed by two different statistical 

procedures, and some values are significant with 

one of these and others are significant with the 

other method. Firstly, why were two tests used 

and secondly, how is the reader to interpret 

significance obtained using two different 

methods on the same dataset?

According to the study director, organ weight data 

are analyzed by parametric procedures using 

Dunnett’s test when data are homogeneous and by 

the Modified t-test when data are nonhomogeneous. Data for each organ parameter 

[absolute wt, relative wt compared to body wt or 

brain wt] is evaluated for homogeneity 

independent of other organs and the appropriate 

statistical test is applied to comparisons of all dose 

groups within that parameter. In this study, 

statistical significance was determined by a non-
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parametric test only once, in the data set for male 

lung wt relative to body wt at terminal sacrifice; 

the relative weight at 20,000mg/m3 was 

significantly higher than controls. The absolute 

lung wt in high dose males was not increased 

significantly. Dr Schlesinger had suggested that 

transformation of data when variances are not 

homogeneous and subsequent use of parametric 

tests throughout could make analysis of all data 

sets more consistent [see response for Section 

2.20.1]. The RG believes that, in lieu of using 

transformed data, the laboratory’ approach for 

evaluating homogeneous and non-homogeneous 

data sets by separate appropriate methods within 

each parameter is a reasonable method for 

determining statistically significant changes in 

each organ. 

Peer Reviewer – Goldsworthy

The potential inhalation toxicity of G-ETBE was 

assessed via whole body exposures to male and 

female rats for 13-weeks followed by a 4 week 

recovery period. In this report, standard 

toxicology neurotoxicity parameters were 

presented (genotoxicity and immunotoxicity 

results were presented in separate reports; 

reviews presented below). The design of the 

study was appropriate and sound and the study 

was properly conducted. The report was clear 

and comprehensive. The deviations noted had no 

effect on the study results or data integrity.

No comments needed

Exposure techniques and procedures were 

adequate to test the potential toxicity of gasolineETBE. Exposure levels of test substance were 

relatively close to target levels although 

differences between measured and nominal 

concentrations were greater than the expected 1:1 

ratio. Although the causes of the differences 

were not determined, it was hypothesized that 

differences may have been due to problems with 

chamber airflow and the IR monitor. Animals 

were exposed to all major components of 

gasoline-ETBE in their expected proper portions. 

Week to week data consistently was noted as 

See response to Dr. Schlesinger comment on 

Section 3.1 Chamber monitoring – IR monitoring.
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well, indicating stability of test substance and 

proper techniques.

Experimental data revealed no effect of G-ETBE 

on survival, feed consumption, clinical observations, or gross abnormalities at necropsy. No 

exposure-related effect was observed on 

functional observation battery or motor activity; 

no effect was observed with ophthalmoscopic 

assessments. There were no exposure-induced 

differences in absolute body weights. The 

20,000 treated female rats exhibited decreased 

body weight changes between exposure weeks 4-

12. There were no observed exposure-related 

effects in coagulation values at 4 weeks and 

terminal intervals. High dosed animals exhibited 

decreased reticulocyte counts at 4 weeks; this 

was not seen at the terminal sacrifice. Similarly, 

various high dose exposure-related differences in 

clinical chemistry values at 4 weeks were not 

present at the terminal intervals. High dosed 

exposure-induced increases in the kidney weights 

of male rats were considered to be the only 

exposure-related toxicological effects. Renal 

microscopic findings in male rats were consistent 

with hydrocarbon-induced nephropathy; this 

species and gender specific alteration is generally 

considered no relevant for human risk 

assessment. I concur with the study conclusions 

and the NOAEL setting of 10,000 mg/m3

exposure level. 

211(b) Research Group Reviewer

EPA has requested that the proportion of 

oxygenates in each gasoline/oxygenate blend be 

added to reports [see BG+EtOH comments, p. 2 

under Immunotoxicity, 6/9/03]. To be accurate 

and consistent across studies, the % oxygenate in 

the starting liquid and % oxygenate in vapor

condensate or at least % oxygenate in vapor 

condensate could be added to the text [2.4 Test 

substance] of each subchronic report. This data 

is available in the Analytical Appendix but 

reviewers do not always consult that section.

The RG has decided that this information will be 

provided in a separate report addressing 

composition and analyses of all test samples.

[Paula, Lorraine – is this the current thinking?]
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13 Week Subchronic Study:

Dr. Schlesinger noted that since the 

Immunotoxicity assay showed positive results for 

BG/ETBE at 10000 and 20000mg/m3, a NOAEL 

of 2000mg/m3should be considered for the 

subchronic study of which the Immunotixicity 

study was a part. [A similar issue arose for the 

positive results in the GFAP assay of BG/EtOH.] 

Assignment of overall LOAELs and NOAELs 

will be determined by the 211(b) workgroup.

A table has been added to Draft 2 summarizing the 

results of all satellite studies so that the reader will 

be fully apprised of NOAELs for different 

endpoints. All satellite studies are supplied as 

Appendices to this report.

Summary:

p.6, 1st paragraph, next to last sentence: major 

components were assayed once per chamber per 

week. Please add “per week”.

Added.

p.6, 2nd paragraph, final sentence: Please add 

that control rats as well as 20000mg/m3 rats were 

examined histopathologically at terminal 

sacrifice, and that Recovery were not evaluated 

histopathologically. 

Added.

p.6, QA requested that variation between 

nominal and analytical concentrations be 

specified. Variations were 4-12%; both values 

were close to or slightly above target 

concentrations.

Not added since the difference was minimal and to 

be consistent with prior reports in this test 

program.

p.7, The change in creatinine kinase is a 

decrease, not increase.

Revised to decrease.

Methods

p.19, 2.5.6 Weight at Initiation of Exposure: 

Please verify that weights for female animals in 

the Immunotoxicology satellite group are the 

same here as in the Immunotoxicology report 

Appendix B. According to QA there is some 

mix-up with these data in that report [item 17, 

Audit dated 1/13/03]

Body weigths reviewed and report appears to be 

correct as is.

p.29, 2.12.5 Feed Consumption: Is it correct to 

assume that feed consumption was calculated for 

each animal and then averaged for the dose 

The reviewer is correct.
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group? See item 21 in QA report [Audit 

1/13/03].

p. 36, 2.16 Immunotoxicity

Line 7: please spell sensitized with a z, rather 

than s. 

Done.

p. 43, 2.20 Statistical Analysis: Report states 

that Mean Clinical pathology values were 

evaluated statistically only on termination data. 

Since when? Statistics in all 211(b) studies were 

run at both wk4 and at termination. Tables 

indicate this as well. Pleas check and correct 

[cited by QA also, Audit 1/13/04].

Revised.

Descriptions of additional neurobehavioral 

analyses, recommended by EPA, will be added to 

this section.

Done.

Results and Discussion

Restate somewhere in the Results that animals 

were treated for a minimum of 65 exposures over 

13 weeks, perhaps at the end of the chamber 

monitoring paragraph [p. 47]. Peer reviewers 

have requested confirmation of the number of 

exposures.

This has been added to the Summary on page 6 

and the M/M on page 15.

p.47, 3.1 Chamber Monitoring: Since QA 

requested that the variation between nominal and 

analytical concentrations be specified in the 

Summary, the information belongs here as well. 

The variations were 4-12%, both values were 

close to or slightly above target concentrations.

Not added since the difference was minimal and to 

be consistent with prior reports in this test 

program.

p.50, 3.7 Neurobehavioral Studies: Any 

changes in interpretation due to application of 

revised statistics requested by EPA will be 

addressed.

Added to report. p.

p.50, 3.8.1 Hematology and Coagulation (Table 

9): Please add the following entries here and, if 

appropriate, in the Summary:

- At week 4, statistically significant (p<0.05) 

increases were seen in MCV for males at 10000 

Added to report.
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and 20000mg/m3; mid dose was slightly higher 

than high dose value and effect was not seen at 

termination.

- Decreased reticulocyte counts similar to those 

seen at week 4 were also present in females 

exposed to 10000 and 20000mg/m3at 

termination but were not statistically significant 

because control values were also lower [cited by 

Peer reviewer, see API submission to EPA, 

7/15/04].

All values were lower at termination than at 

interim interval – this is normal change in 

maturing rats. Therefore, no discussion added to

the report.

p.50, last sentence: Add “no significant exposure 

related differences...........”

Added to report.

p. 51, 3.8.3 Clinical Chemistry (Table 11): line 

7 [2nd sentence]. Please correct. Change in 

creatinine kinase over three doses is a decrease, 

not an increase that is statistically significant at 

20000mg/m3.

Revised report.

Chloride values were statistically significantly 

increased (p<0.01) at wk 4 in females but the 

increase was the same over 3 doses and was not 

observed at termination. Although the data 

indicates a plateau effect and was not seen at wk 

13, a significance level of p<0.01 should be 

mentioned and probably discounted as not 

biologically significant.

Added to report.

211(b) Research Group QA/QC Reviewer

A raw data and draft final report review was 

performed to assure compliance with GLP 

regulations (Part 79.60). One hundred percent of 

the text and summary tables of the report were 

verified and at least 10 percent of individual data 

were verified. The following were reviewed and 

inspected:

1. Protocol

2. Draft Final Report dated June 19, 2002

3. Red Spiral Binder labeled A-12, 00-6129, 

Background – No comments needed
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Gasoline ETBE Vapor Condensate, A-12, 00-

6129, Gasoline ETBE Vapor Condensate: a 13-

Week Whole Body Inhalation Toxicity Study in 

Rats With Neurotoxicity Assessments and 4-

Week In Vivo Genotoxicity and Immunotoxicity 

Assessments, Volume II, Book 17 of 18 Section: 

Charcoal Tube Monitoring Record.

4. Clear Binder labeled Study Title: Gasoline 

ETBE Vapour Condensate, A-12, 00-6129, 

Gasoline Tame Vapor Condensate A 13 Week 

Whole-Body Inhalation Study in Rats with 

Neurotixicity Assessment and 4-Week In Vivo 

Genotocixity and Immunotoxicity Assessment, 

Study Director: Gary M. Hoffman, B.A., 

D.A.B.T; Scientist: Samuel R.Onanuga, M.S., 

Associate Director; Tox-Support Chemistry: Kay 

Saladdin, B., Book: 1 of 2

Sections: Exposures, Method Development, 

Invalid—Raw Data

5. Clear Binder labeled Study Title: Gasoline 

ETBE Vapour Condensate, A-12, 00-6129, 

Gasoline Tame Vapor Condensate A 13 WholeBody Inhalation Study in Rats with Neurotixicity 

Assessment and 4-Week In Vivo Genotocixity 

and Immunotoxicity Assessment, Study Director: 

Gary M. Hoffman, B.A., DABT, Scientists: 

Samuel R. Onanuga, M.S., Associate Director, 

Tox-Support Chemistry: Kay Saladdin, B.S. 

Book 1 of 2

Sections: Abbreviations, Information, Protocol, 

Analytical Procedure, Pipette Calibration, 

Method validation, Pretest Trials, Exposures

6. Clear Binder labeled A-12, 00-6129 

Gasoline ETBE Vapor Condensate: A 13-Week 

Whole-Body Inhalation Toxicity Study in Rats 

With Neurotoxicity Assessments and 4-Week In 

Vivo Genotoxicity and Immunotoxicity 

Assessments Volume II Box 3 of 8

Section: Charcoal Tubes

7. Clear Binder labeled A-12, 00-6129, 
211(b) Toxicology Research Group

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Gasoline ETBE Vapor Condensate: A 13-Week 

Whole-Body Inhalation Toxicity Study in Rats 

With Neurotoxicity Assessments and 4-Week In 

Vivo Genotoxicity and Immunotoxicity 

Assessments Volume II Book 2 of 18

Sections 1 Body wts, food wts and Clinical 

Observations

8. Clear Binder labeled A-12, 00-6129, 

Gasoline ETBE Vapor Condensate: A 13-Week 

Whole-Body Inhalation Toxicity Study in Rats 

With Neurotoxicity Assessments and 4-Week In 

Vivo Genotoxicity and Immunotoxicity 

Assessments Volume II Book 2 of 18

Sections: Computer Raw Data Changes and 

Additions Edits, Daily Environment and Viability 

Records, Clinical Observation Study List, 

Computer Sort

9. Clear Binder labeled 00-6129, Clinical 

Pathology Stats •Hematology• Coagulation •

Clinical Chemistry

FOB stats• Foot Landing Splay •Grip Strength •

10. Clear Binder labeled Clinical Pathology 

Raw Data, Table of Contents, Sponsor Code: A12, Study 00-6129, Book 1 of 1, Volume:

Sections:

I. Signature Page, Raw Data correction 

codes, Protocol parameter page, Memos

II. Transfer sheets

III. Clinical Chemistry

IV. Coagulation

11. Clear Binder labeled A-12, 00-6129, 

Necropsy Data Book 1 of 1

12. Chamber Monitoring Records

13. Test Material Usage and Dispensing Logs

14. Test Material Receipt Records
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15. GFAP Raw Data

16. Eye Exam Observations

17. Motor Activity Data

18. FOB Observations

19. Animal receipt Records

The following items are comments or require 

further consideration:

1. Page 2, Compliance Statement:

The sponsor also needs to sign a compliance 

statement. It can be a separate one from the 

Testing Facility’s but there must be one signed 

by the sponsor. It should also include those nonGLP compliant issues that the Testing Facility 

has indicated. 

Added to report.

2. Compliance Statement: Since it is the 

sponsor’s responsibility to maintain the method 

of synthesis, fabrication, or derivation of the test 

fuel, and this had not been completed, it must be 

included in the sponsor’s compliance statement

Added to report.

3. The status of the GFAP laboratory 

concerning GLP compliance should also be 

included in the GLP compliance statement signed 

by the sponsor. A list of non-GLP compliant 

issues is being prepared for the GFAP report. 

These items must be included in the statement

Added to report.

4. Page 4, Quality Assurance Statement: Since 

HLS has been inspected all of the subcontractors 

(except CDC) these facility inspections should be 

listed on 

The inspections were generic and not study 

specific and therefore will not be listed on the QA 

Statement.

5. The date that the QA report of the report 

draft and study data was reported to the Study 

Director and management needs to be added to 

the QA Statement.

Added to report.
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6. Page 6, Summary: When indicating the 

different reports (various places in the report), 

they should be noted as “appended” to this 

report. They are stand-alone reports, but since 

they were requirements of the protocol, they need 

to be reported with the rest of the study.

Added to report.

7. Page 6, Summary, first paragraph, last 

sentence: Major components were assayed once 

per chamber, per week.

Added to report.

8. Page 6, Summary, second paragraph, next 

to last sentence: Histopathological evaluations of 

all selected or prescribed tissues were 

conducted on all control and 20,000 mg/m

3

exposed.......Lungs and gross lesions were also 

examined for mid dose animals at the Terminal 

interval. Recovery animals received..........

Added to report.

9. Page 6, Summary, third paragraph: are the 

values reported for chamber concentrations grand 

means and standard deviations for both 

chambers? They appear to be the mean of the 

means and standard deviations of the two 

chambers. Please clarify.

10. Page 6, Summary, third paragraph, third 

sentence: Some idea of the extent of the variation 

between nominal and measured concentrations 

should be given.

The values reported for chamber concentrations 

are grand means and standard deviations for both 

chambers as explained on page 48.

Not added since the difference was minimal and to 

be consistent with prior reports in this test 

program.

11. Page 7, Summary, third paragraph, 

reporting of body weight findings: Due to the 

manner in which the body weight changes were 

calculated (always the change from 0 to all 

intervals) it is difficult to tell if there were body 

weight changes at one interval or all of them. If 

they had been reported by interval, 0-1, 1-2, 2-3, 

etc. one may see that there were no differences 

throughout the 12 weeks.

Not practical due to computer system limitations.

12. Page 7, Summary, paragraph 4, second 

sentence: it appears there was a decrease in 

Revised report.
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creatine kinase values instead of an increase. 

Please clarify.

13. Page 8, Summary second paragraph, first 

sentence: The results section indicates this 

sentence indicates that no gross abnormalities 

clearly related to test substance.... Please clarify.

Revised report to clarify ‘exposure-dependent 

incidence of bilateral renal foci of red 

discoloration’.

14. Page 17, Date Received: There is a record 

of cylinders being received on 9/10/01. Were 

these empty cylinders? Please clarify.

Cylinders were only received on 17Sept01 and this 

included 4 large cylinders full of test substance and 

20 small empty cylinders.

15. Page 17, Expiration Date: It should be 

indicated that the test substance stability was 

tested concurrently with the study and the results 

showed it to be stable for the duration of the 

study. (See Appendix S?)

Added to report.

16. Page 18, Supplier: Are Kingston and 

Stoneridge, NY the same place. Animal records

indicated Stoneridge, NY.

These are equivalent Charles River locations.

17. Page 19, Weight at Initiation of Exposures 

(Grams): The values for Immunotoxicity will 

have to be corrected when these values are 

corrected in the report tables.

BW’s reviewed and appear to be correct as is.

18. Page 20, Veterinary Care: It seems that any 

conditions requiring veterinary care need to be 

reported whether they are felt to be non-test 

substance-related or not.

No change required, information is maintained in 

raw data if needed.

19. Page 23, Environmental Conditions: Some 

indication of the extent of the excursion should 

be given, ie, only occurred on 11 days in one of 

the high-dose chambers, etc.

Temperature excursions were minimal (+2C) and 

need not be detailed further.

20. Page 28, Ophthalmoscopic examination: 

Shouldn’t it be noted that animals with ocular 

findings were not used for study.

This was noted in Section 2.6. Page 19

21. Page 29, calculation: is the denominator the 

body weight (kg) of the group or the individual?

The denominator is the body weight (kg) of the 

individual and then the values are averaged.
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22. Page 32, Relative Humidity: Please verify 

the actual range, I found 40-64% to be the range.

Data checked and range is correct as reported.

23. Page 36, Micronucleus Evaluations: It is 

indicated that the positive control animals were 

sacrificed with CO2, but nothing for the test and 

control-treated inhalation/exsanguinations.

These are the same animals as for the SCE 

evaluation as explained on prior page and their 

disposition was explained there.

24. Page 42, GFAP Evaluations: It is indicated 

that animals were sacrificed with CO2 inhalation, 

but I believe they were anesthetized with CO2 

and sacrificed by decapitation. Please comment.

OK as is – they were killed by CO2 and then 

decapitated immediately for tissue collection.

25. In the report test, Page 43, Section 2.20 

Statistical Analysis states that mean clinical 

pathology values “only run on term”, but mean 

values were run at 4 weeks and term.

Report corrected.

26. Page 51, Clinical Chemistry, second 

sentence: It appears there was a decrease in 

creatine kinase values, not increase. Please 

verify,

Report corrected.

27. All pages in the tables and appendices do 

not contain the Sponsor No. 211-ETBE-S. The 

hematology, coagulation and clinical chemistry 

individual appendices and summary tables should 

be page numbered 

Not done for previous studies in this series.

Full report will be paginated for final report.

28. Table 8, Summary of Functional 

Observational Battery: Group 3, Forelimb Grip 

Strength, Trial 2, Pretest Interval. Please verify 

the mean and standard deviation. I get 

845.5±149.8.

Table 9 has been revised to include mean of the 2 

trials (grip strength and foot splay). All averages 

have been recalculated accordingly.

29. In Table 2, Page 6 and 7, data summaries 

appear to be from the satellite groups for this 

study. Individual data was not included in 

Appendix B to support these summary data.

Pages were misplaced and have been moved.

30. Table 7, Motor Activity Values, Summary 

of Statistical Analyses: Second sentence should 

indicate that there was a recovery period only for 

certain animals.

This entire Table 7 has been replaced by the new 

statistical report from the EMBSI statistician Mark 

Nicolich. New report included in 2nd draft.
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31. In Table 11, Week 4, Group 3M 10,000 

mg/m

3

, the statistics were performed with an n of 

9; the mean was 0.07889 with a standard 

deviation of 0.0232. Table 11 reports an n of 10, 

mean 0.07 with an SD of 0.0233. The statistics 

should be rerun using all 10 values. The values 

in Table 11 appear to be correct.

The statistics were re-run with all 10 animals in 

group 3 males.

32. Different Ophthalmologists examined the

animals pretest and at 13 weeks. This should be 

indicated in the ophthalmology report (.e, which 

data was observed by each Ophthalmologist. It 

does appear in the listing of personnel.

This is shown in Table 3 by the summary letters 

from the 2 different examiners.

33. Table 14, Lesion Incidence Summary with 

Expanded Severity Levels: The preface to this 

table does not include an explanation of NAD. 

Please verify that NAD is defined somewhere in 

the report.

Added to preface page.

34. Table 14, Lesion Incidence Summary with 

Expanded Severity Levels: For tissues that were 

examined and found normal, why is there no 

indication of normalcy on the summary table? 

Should NAD be entered where there are no 

findings?

NAD is defined on the Preface. Only if there are 

findings in some animals does NAD print for the 

rest. No change can be made to the manner in 

which the program operates. No change to report. 

35. Table 14, Appendix M: There is a comment 

of Atelactasis for several animals. Is this 

something that should be included in the 

summary tables, even though it is probably an 

artifact? It is not included.

Comments are not included on summary tables and 

are limited to the individual data pages. No 

change to report.

36. Appendix A. Pretest particle size results 

need to be reported.

Data has been added to Appendix Q.

37. Appendix A, Chamber Monitoring Results: 

For the Particle Size Determinations, Total Mass 

Concentration, the standard deviation is printed 

as 0.0. Actual values need to be included.

Number of decimal places in the mean GSD has 

been revised to match mean values presented in the 

same table.

38. Appendix A, Chamber IIIB, the nominal 

value for Nov 7 should be 10.200, I believe. 

Please verify.

CMR revised (all affected satellite reports revised 

as well).
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39. Appendix A. Chamber IIA, second value 

for Dec. 11. Please verify that the value is 2240 

and not 1510 mg/m3.

The value of 2240 is correct (this represents an 

average of the 1st and 2nd duplicate sample and was 

calculated according to SOP).

40. In Appendix B, Page 10, Animal 5062—

The edit change on Day 27 for this animal and 

Animal 4061 should be reviewed. The finding 

“red nasal discharge” that was observed for 

Animal 4062 (seen previous week and following 

week) was edited out and two findings that were 

recorded for Animal 4061 were added to Animal 

4062. Should all three findings be present for 

Animal 4062?

Raw data corrections reviewed and it appears to be 

intentional that red nasal was removed as an 

incorrect entry. No change to report.

41. In Appendix D, individual data is reported 

from Week-1 to Week 17. Appendix E reports 

weight change from Week 0 to Week 17. Should 

the weight change from Week-1 to Start of Study 

(Week 0) also be reported in Appendix E?

No - body weight change is calculated and 

presented from baseline (Week 0). No change to 

report.

42 The Preface, Page 5 of Appendix H for 

abnormal movements does not match the key in 

the data notebooks. A clarification to the raw 

data key is necessary to explain convulsions, 

tremors, or fasciculation as abnormal movements 

in order to match the key as being reported in 

Appendix H.

Key in data lists “Abnormal Motor Movements”; 

convulsions, tremors, or fasciculation are listed on 

data pages under “Motor Movement” heading; data 

appears to be clear as is.

43. Appendix H, Individual Functional Obs, 

Battery Ev.: Several animals were noted in the 

data with tremors when handled. This is not 

reported, (animal numbers, 4572, 3568, 1578, 

3565).

Table has been up-dated.

44. In Appendix J, Group 3, Females, PT value 

-- The individual values for Animals 3553 and 

3554 records “EM” meaning equipment 

malfunction, results not valid. The raw data 

records no plasma. An explanation is needed to 

clarify the “no plasma” recording versus 

“equipment malfunction.”

Data indicates QNS due to EM, therefore reporting 

the value as EM appears to be accurate. No 

change to report.

45. In the clinical chemistry values, Appendix The reported value of 0.0 is OK and will be 
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K, Week 4, Animal 3054—The reported values 

for TBIL – 0.03 and DBIL 0.04. This results in n 

IBIL value of -0.01, but the table reports 0.0 as 

the value. Should this value be footnoted and 

explained?

footnoted with explanation that a negative value is 

not possible. Footnote “Result was -0.01 but 

presented as 0.00 since a negative biological value 

not considered realistic” added to report.

46. Appendix L needs to be page numbered. Report will be paginated for finalization.

47. Appendix S, Analytical Report: The table 

on Page 5 of the analytical report does not record 

that samples were collected on 12-14-01.

Added to the report.

48. Appendix S, Analytical Report: The 

required signatories as listed on Page 2 of the 

analytical report should sign the analytical report.

This will be done when the final report is prepared.

49. Appendix S, Analytical Report: The table 

on Page 7 of the analytical report records ND for 

the total from columns Samples Group 1A and 

Samples Group 1B. All other tables in this report

record 0.00 for the total of these samples. The 

0.00 should be used for consistency.;

Corrected

50. Appendix S, Analytical Report: The four 

figures in the analytical report should document 

that the chromatograms were obtained from 

samples collected during Exposure 3.

Corrected

51. Appendix S, Analytical Report: In Figures 

II, III and IV of the analytical report, 15 peaks 

were labeled. The peaks for 2,3 

dimethylpentane, 2, 4 dimethylpentane, 2 

methylpentane and trans-2-pentane are not 

labeled.

Printout is accurate but not all peaks are labelled 

on the printouts due to printing constraints where 

peaks, such as those noted in the comment, are 

compressed into each other.

52. Appendix S, Analytical Report: In Figure 

III of the analytical report, the response time for 

the peaks 2,3-dimethybutane and benzene do not 

agree with the values as reported on the raw data 

chromatograms.

Printout is accurate but not all peaks are clearly 

labelled on the printouts due to printing constraints 

where peaks, such as those noted in the comment, 

are compressed into each other.

53. Appendix S, Analytical Report: In Figure 

IV of the analytical report, the response time for 

the peaks 2,3-dimethylbutine, n-hexane, and 

Printout is accurate but not all peaks are clearly 

labelled on the printouts due to printing constraints 

where peaks, such as those noted in the comment, 
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benzene do not agree with the values as reported 

on the raw data chromatograms.

are compressed into each other.

54. Sister Chromatid Exchange Assay Report, 

Page 9: The last line contains a redundancy. I 

believe the 68 hours can be deleted.

OK as is- this is correct per protocol.

55. Sister Chromatid Exchange Assay Report, 

Page 9: Can it be verified that the sodium 

heparin tubes stability had not expired?

I do not have an answer to this query from 

Bioreliance but assume that they would not have 

used expired sampling tubes.

56. Sister Chromatid Exchange Assay, Page 9: 

The lot number for Cyclophosphamide, 

108H0568 is the same from the two previous 

studies, but they all have had different expiration 

dates. Could the same lot have different 

expiration dates?

Yes – the expiry date was extended by the 

manufacturer.

57. Sister Chromatid Exchange Assay Report, 

Page 19: The mean SCEs per cell for animal 

number 5052 should be 22.2 not 21.7, I believe. 

Please verify.

Report corrected.

58. Sister Chromatid Exchange Assay Report, 

Page 20: The AGT per group value for Group 1 

appears to be 26.6 not 26. Please verify.

Report corrected.

59. Immunological Evaluation Assay Report: 

The body weights, body weights gains, food 

consumptions and resulting mean tables have 

animals mixed up. All groups are represented 

under any group number. This means also that 

the means for groups is not correct. These 

animals need to be rearranged and data needs to 

be in the correct table.

Issue corrected by HLS IT and pages have been 

re-run.

60. It also raises the concern that this should 

not have happened. The computer should not let 

animals of different groups be summarized 

together. Please comment.

Per HLS IT, 1 file was not copied to a particular 

environment with the rest of the data; issue has 

been rectified.

61. Immunological Evaluation Assay Report, 

Page 9, Table ES-1: It is assumed that the 

Yes – the maximum effects were seen at the 

middle exposure level.
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column titled “Maximum Effect” is there to 

indicate that the largest effect was seen in the 

10,000 and 10,000 mg/m3 levels the reader can 

gather this.

62. Immunological Evaluation Assay Report, 

Page 11, second sentence starting with 

Cyclophosphamide: This sentence is a repeat of 

the first one beginning with Cyclophosphamide.

Corrected

63. Immunological Evaluation Assay 

Report, Page 13, Terminal Body Weights and 

Organ Weights: It only gives a weight for the 

control animals. Shouldn’t it compare the 

controls to the dosed groups? (Note: This mean 

may change when body weight tables are 

corrected).

Sentence listing only control weights deleted. The 

treated groups were statistically compared to the

vehicle control group as per protocol and this 

report.

64. Immunological Evaluation Assay Report, 

Page 16, second sentence: The decrease in the 

IgM-antibody-forming cell response was not 

dose-related when evaluated as total spleen 

activity, so the term dose-related should be 

deleted.

The term has been deleted.

65. Immunological Evaluation Assay Report, 

Page 17, second sentence: This sentence should 

probably indicate the “immune” functional 

ability of the animals.......

The sentence has been corrected and it now 

indicates “humoral immune” functional ability of 

the animals.

66. Immunological Evaluation Assay Report, 

Page 17, last sentence: Shouldn’t it be indicated 

that there was an adversely affected humoral 

immune response of female SD rats “at levels of 

10,000 and 20,000 mg/m3?” And that there was 

a no effect level? This should also be indicated 

on page 8, the Executive Summary.

This has been corrected in both locations.

67. Immunological Evaluation Assay Report, 

Page 2, GLP compliance statement: The 

sentence indicating that the “method used for 

antibody-forming cell assay had not been 

formally validated” needs to be clarified. It was 

my understanding that the assay was validated 

Corrected.
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and that producing a formal written procedure for 

doing so was ongoing. Please indicate this in the 

GLP compliance statement.

68. GFAP Report: It is indicated that the “spirit 

of GLPs” were followed. It would be useful to 

indicate where the study fell short of full GLP 

compliance.

Chris Sexsmith to prepare a listing of deficiencies.

69. GFAP Report, QA Statement: It is 

indicated that the findings of QA reports were 

reported to the Study Director and Management. 

Was this Gary Hoffman, the Study Director and 

His Management at HLS? That is the way it 

should be as there can only be one Study Director 

in a Study.

Gary Hoffman is Study Director.

70. GFAP Report, Page 8, second sentence: 

There is not a Two-Generation study to be done 

with ETBE. It will only be a One-Generation 

study. As well the next sentence should indicate 

that this study was to use the GFAP assay for 

assessing the effects in a subchronic study. The 

in-life animal data should also be referenced.

ETBE changed to MTBE.

71. GFAP Report, Page 17, section D, Typical 

Results: These data need to be further identified. 

Are these comparable strains, ages, sexes, etc.? 

These data actually have to be trackable to a 

study. Are there data available/compiled to 

verify this Figure?

Changed first sentence to: “Typical GFAP assay 

values obtained at this laboratory for different 

regions of the rat brain are presented in Fig. 2 (i.e. 

historical data).” And a citation for these results is 

now mentioned in the Figure Legend (p. 21) and 

reference section (Martin and O’Callaghan, 1995).

72. GFAP Report: For Tables 5 and 6, the group 

4 female and male pituitary values are based only 

on one animal so should probably not be included 

in the mean table.

Pituitary data removed from these summary tables.

73. GFAP Report, Tables 5 and 6: Values for 

Cortex in Group 4 males and females appear to 

be switched or incorrect. Male value should be 

1.72±0.07 and the female value should be 

1.76±0.06. As well, the cortex value for the 

Group 1 females SEM should be 0.08, and the 

SEM for the Hippocampus value for the Group 3 

males should be 0.22. Please verify.

Corrected mean values. SEM values are computer 

generated so okay as is.
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74. GFAP Report, Table 7, The Pituitary values 

reported for the group 4 males and females are 

not correct.

Values are corrected.

75. GFAP Study: It appears that there was a 

large coefficient of variation in the data and that 

is the reason why many sample analyses were 

repeated. There needs to be a detailed 

explanation of this in the data, and repeat values 

need to be footnoted as such. Actual values 

reported need to be clearly indicated in the data.

Usual laboratory experimental procedures; 

Repeated GFAP on sample if %CV is > 15% then 

include the values that are <15% CV in the report. 

No comment necessary.

76. Micronucleus Report, Page 15, second 

paragraph: It appears that the group mean value 

of 2.4 is within the group mean historical control 

range. Please verify.

The highest value included for group mean values 

in the vehicle historical control data is 2.1, 

therefore the value of 2.4 is outside the vehicle 

historical control range and the report is correct.

77. Micronucleus Report, Page 16: Should the 

conclusion indicate that the possible increase in 

frequency of micronuclei was only in females?

No, we don't think the conclusion should be 

changed, as values for all females were within the 

individual historical vehicle control range and the 

increases observed in the group mean incidences 

were not dose related and not sex specific.

78. Pathology Report: For all animals were 

only gross observations were reviewed 

microscopically, the pathology table indicates, 

“Tissues w/o comment under gross observations 

were within normal limits,” and “The following 

tissues were unremarkable microscopically, no 

tissues examined”, yet the tissues with gross 

lesions were examined. It is confusing. Please 

clarify.

This refers to all other tissues aside from the ones 

with gross lesions. No clarification needed.

DATA Findings:

1. In the GFAP data book, it is indicated that 

Christine Mason is the API Principal 

Investigator. This should be corrected.

Data clarified.

2. There appear to be numerous tissues 

missing and damaged from the Group 1 animals. 

Is there an explanation for this? Many recuts 

were requested and sometimes tissues were not 

We have reviewed the raw data and do not find the 

number of recuts or damaged tissues to be 

excessive compared to other studies from this prior 

timeframe or our current timeframe.
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Inhalation Toxicity Report

Comments Action/Response Page #

found. Please comment.

3. Appendix S, Analytical Report Data: The 

titles on the raw data notebooks for the analytical 

analysis of the charcoal tubes should be 

corrected: the titles states TAME and the study 

is ETBE, several words in the name of the study 

were misspelled, and the second notebook should 

be labeled Book 2 of 2, not 1 of 2 as the first 

notebook was labeled.

Corrected.

4. Analytical Report Data: Freezer stability 

for the charcoal tubes is necessary in the method 

validation to support the data from the charcoal 

tubes being frozen after collection and before 

extraction (one day and 13 days storage).

Freezer stability was not conducted and will be 

noted on the GLP Compliance Statement.

5. Analytical Report Data: The charcoal tubes 

collected on 12-14-01 were analyzed and the data 

was rejected. New samples were collected on 

12-17-01 and the results from this analysis were 

reported. Documentation is needed in the data to 

explain why the data from 12-14-01 was rejected 

and the location of any data that was obtained 

during the analyses of the 12-14-01 samples. 

This rejected data should be included in the 

notebook with the explanation of the situation. 

The data states “instrument problems and 

response” but the documentation does not record 

what the instrument problems were and what was 

done to correct the situation.

No samples were injected due to an instrument 

error. The details of the instrument error were not 

documented.

6. Analytical Report Data: In the 

analytical raw data for Exposure 35, the sequence 

of samples records incorrect sampling numbers. 

The sample number states 12 samples, but 14 

samples were analyzed. Sample 5 is listed 

incorrectly three times in the sequence.

Correct as is. The sequence was manually 

corrected.

7. In the clinical chemistry data a note should 

be made to clarify that for the GGT parameter –

when the results is a –1L alert, -2L alert, or -3L 

alert, the value is reported as 0 and the sample is 

not rerun. All other instrument flags result in the 

A memo has been added that explains the SOP 

requirements for reporting values below the assay 

linear limits.
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Comments Action/Response Page #

sample being rerun. This procedure should be 

clarified.

8. In a discussion with the Supervisor of 

Clinical Pathology, the SOP CP1.2.22 details the 

requirements for performing and reporting a 

manual differential versus the automated reading 

for the differential. The supervisor stated that the 

technician would review the raw data and if the 

% Monocytes is greater than 7, or the % 

Eosinophils is greater than 5, or the % 

Leucocytes is greater than 5, a manual 

differential count would be performed. If the 

manual differential count confirms the automated 

values, the automated values would be reported. 

If the manual values were different than the 

automated values, the manual values would be 

reported.

Correct as is. 

SOP procedures indicated that this is correct.

The problem of performing statistical 

calculations using the absolute values obtained 

from the manual differential with the automated 

values was also discussed. The supervisor stated 

she would consider removing the absolute values 

from a manual differential from the statistics 

since the number of cells observed are different. 

But the % values should be included in the 

statistics since the number of cells observed is 

used in the calculation of the percentages.

The SOP in place at the time of analysis required 

that the manual differential count and the 

calculated absolute counts be reported. The LUC 

(U%) are counted by the Advia but manual 

differential does not count LUCs, therefore LUCs 

were reported as 0.0.

9. The one-generation protocol for Baseline 

Gasoline and TAME is enclosed in the pharmacy 

file for this study.

Protocol has since been removed.

10. Test Material Inventory Records: Large 

Container number 1 should have 235,348 g not 

224,063. Please verify.

Data has been corrected.

11. Test Material Inventory Records: There are 

records indicating two #1 containers that are 

empty. As well, a sheet for study 6129/4203 

indicates sample #1 returned to sponsor on 

3/15/02, but container #1 was returned to 

Chevron on 12/7/01. Please clarify.

We have reviewed the records and they clearly 

indicate that only 1 container #1 of G/ETBE was 

returned on 7Dec01. The 15March02 shipment 

was for G/ETOH not for G/ETBE.
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Comments Action/Response Page #

12. Lactose Dehydrogenase values, particularly 

for the control animals, appear very irregular. 

Are there any explanations for this? 

It is known that LDH levels in rat are highly 

variable. A review of the data did not indicate any 

unusual findings with the assay itself. QC results 

were within range. Sample processing record did 

not indicate any anomalies either. The handling of 

the animals during bleeding may have contributed 

to the irregular readings.

Micronucleus Satellite Report

Comments Action/Response Page #

Peer Reviewer - Schlesinger

In the statistical section, it is noted that the results 

of the trend test with Group 4 excluded was not 

significant, and this was likely due to the low 

power of the test with available data. If power is 

low, then why use the test at all?

Because of the equivocal nature of the data from 

the first set of slides, a second set of slides was 

evaluated and appropriate statistical evaluations 

performed.

The use of both p> designations depending upon 

the phrasing of the statement adds a certain level 

of potential confusion for the reader. All 

statements should be stated as p< for consistency 

and to avoid any confusion or misreading of the 

results.

The use of <p only is the convention in Draft 2

Table 1 – There should be some error terms (SE 

or SD) provided with the group means for the % 

and incidence datasets. This may help in 

understanding why a value of 2.7 in the incidence 

column of the combined gender table is not 

significantly different from control when a value 

of 2.4 is.

Error terms have been included in Draft 2 for the 

first and second set of data presented

It is also unnecessary to indicate that p<0.001 is 

highly significant. The p values speak for 

themselves.

Acknowledged

Peer Reviewer – Goldsworthy

Study was properly conducted and scientifically 

sound; no study deviations were noted. Positive 

and negative controls performed as expected and 

within historical range. Gasoline-ETBE did not 

cause any increases in the incidence of 

micronucleated mature erythrocytes and did not 

A second set of slides from the micronucleus 

assay was read and the entire report revised to 

reflect the additional data. Although there was an 

apparent increase in group mean micronucleated 

immature erythrocytes (mie) with increasing 

concentrations in the second set of slides, none of 
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cause any decreases in the proportion of immature 

erythrocytes. Group mean values from both the 

intermediate and high exposure level treatment 

groups exhibited some increase over negative 

control values and were outside the group mean 

historical control range. Various statistical 

analyses on pooled data from both sexes and from 

males and females separately exhibited some 

significant increases. Changes were, however, not 

of high magnitude nor consistently exposure 

concentration related. Thus, I concur with the 

report that Gasoline ETBE Vapor Condensate 

may cause an increase in frequency of 

micronuclei in immature erythrocytes. No 

evidence of bone marrow toxicity was observed.

the values from animals exposed to BG+ETBE 

Vapor condensate were statistically significant 

compared to negative controls. The report has 

been re-written as draft 2 to include these data.

Editorial:

Page 8, replace “gasoline-TAME” with 

“Gasoline-ETBE”

Report corrected. Page 9

211 (b) Research Group Reviewer

The initial draft report [June 20, 2002] gave 

equivocal results in which only mid-dose female 

rats showed a statistically significant increase in 

micronucleated immature erythrocytes (mie) 

when analyzed by the permutation test; no 

significant effects were seen in males or for 

combined sexes. A linear-by-linear trend test was 

significant for combined sexes at 20000mg/m3

even though the mie incidence was lower than at 

10000mg/m3– this significant trend disappeared 

when the high dose was excluded in the stepdown analysis. No significant trends were seen 

for males or females evaluated separately.

The second set of slides from the same animals 

was evaluated and these data along with the 

original set comprise the second draft report 

discussed here. 

A second set of slides from the micronucleus 

assay was read and the entire report revised to 

reflect the additional data. Although there was an 

apparent increase in group mean micronucleated 

immature erythrocytes (mie) with increasing 

concentrations in the second set of slides, none of 

the values from animals exposed to BG+ETBE 

Vapor condensate were statistically significant 

compared to negative controls. The report has 

been re-written as draft 2 to include these data

General comments from peer reviewers on first 

report: Dr Schlesinger requested the addition of 

error terms [SE or SD] to data sets and also 

requested that only p< terms be used in discussion 

and p> terms be deleted for clarity.

Corrections have been made.
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Comments Action/Response Page #

p. 8, Summary: 5th paragraph – Test material is 

Gasoline ETBE, not Gasoline TAME.

Corrected.

Please move paragraph 7 to above paragraph 6 

and rewrite slightly as:

“In slide set 2, although there was an apparent 

increase in the group mean mie with increasing 

concentration.......etc... The statistical 

significance seen in slide set 1 data was not 

reproduced in slide set 2.” 

Revised.

The next paragraph is the explanation for 

concluding the results of slide set 1 and combined 

1 &2 results as not biologically significant. To 

the 3rditem in this paragraph add, “The trend test 

for combined sexes was significant...

Revised.

p. 17, Results:

Tables: Rearrange the tables so that the table of 

combined sets 1 & 2, currently Table 1 becomes 

Table 5 to coincide with the order of discussion in 

the results. The combined analyses are not 

discussed until p. 19. Thus, Slide set 1 Statistical 

analysis becomes Table 1, Individual animal data 

for slide set 1 becomes Table 2; 

Slide set 2 Statistical analysis becomes Table 3 

and individual animal data for slide set 2 becomes 

Table 4.

Revised.

p.17, 1st paragraph: Change table designations. 

Individual animal data is Table 2, not 3

4

th paragraph: Change table designations. 

Statistical analysis is Table 1, not 2.

7

th paragraph, [Permutation or Wilcoxon Test]: 

Doses units are mg/m3, not ppm. Thus 

10000mg/m3, not 10000ppm

Revised.

Revised.

p 18, 1st paragraph: Change table designations. 

Individual animal data is Table 4, not 5

4th paragraph: Change table designations. 

Revised.

Revised.
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Micronucleus Satellite Report

Comments Action/Response Page #

Statistical analysis is Table 3, not 4.

2nd paragraph: doses are reported as mg/m3, not 

ppm.

Revised.

p.19, 2nd and 6th paragraphs: doses are reported 

as mg/m3, not ppm

3rd paragraph: Statistical analysis table change 

to Table 5, not 1.

Revised.

Revised.

p.19, 2nd and 6th paragraphs: doses are reported 

as mg/m3, not ppm

3rd paragraph: Statistical analysis table change 

to Table 5, not 1.

Revised.

Revised.

p.20, 1st paragraph : Change 10000ppm to 

10000mg/m3

2nd paragraph: Please add “A significant linear 

trend was not seen when sexes were analyzed 

separately”.

5th paragraph describes the results of slide set 2, 

doesn’t it? Shouldn’t slide set 2 be added to the 

sentence? Suggest that this paragraph be moved 

to 3rd paragraph position before the discussion of 

“One male animal from Slide set 2 showed an 

individual value (7) outside the historical control 

range.”

In the paragraph describing the reasons for no 

biological significance, item 3; add “The trend 

test for combined sexes was significant for Slide 

set 1....etc..”

Revised.

Revised.

Revised.

Revised.
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Sister Chromatid Exchange Satellite Report

Comments Action/Response Page #

Peer Reviewer - Schlesinger

Methods

The statistical analysis used is not adequately 

described. The Dunnett’s test is a post-hoc test 

that usually follows an Analysis of Variance 

procedure. This is not indicated in the report.

At the request of EPA, a trend test has been added 

to SCE data analysis. ANOVA was performed 

prior to the application of Dunnett’s test. 

Revisions appear in Draft 2.

Furthermore, the paragraph describing the criteria 

for a positive conclusion is not clear. The 

investigators considered that a test substance was 

positive if an “...exposure-level responsive and 

statistically significant increase is observed over a 

minimum of two exposure levels.” Firstly, how 

was the relationship between exposure concentration and response assessed? There is no indication of any statistical analysis for any trend.

Secondly, it is not necessarily valid to conclude 

that the lack of an exposure level related trend 

indicates that results should be noted as equivocal. 

There can be various reasons why effects at 

different levels could be similar in magnitude but 

biologically “real”.

Where responses at 2 sequential doses are similar 

and statistically significant suggesting a threshold 

for expression, the investigator would likely judge 

the response to be positive. However, if positive 

doses are widely separated and/or values are 

higher at the lower dose in the absence of toxicity, 

then the study could be considered equivocal. 

The SCE assay was developed as an indicator of 

DNA perturbation with possible application for 

identifying potential for chromosome or other 

genetic damage. Criteria for positive responses 

developed by many investigators evaluating 

numerous assays were designed to be sufficiently 

rigorous to minimize false positive and negative 

results in relation to other in vivo genetic assays 

and to strengthen predictability and reliability. 

The designation of “suspect” when statistical 

significance was observed only at the highest 

dose, and “equivocal” when positive results are 

observed at two doses in the absence of an 

exposure response trend contributed to improving 

this reliability by ranking the relevance of 

responses.

Results & Discussion

The actual results are presented at the end of the 

last paragraph of this section

There is no discussion.

No comments

Furthermore, the 1st paragraph of Results should 

be in the introduction while all of the other 

paragraphs until the last half of the last paragraph 

in Results should be in the Methods section. 

Comment has been noted but to be consistent with 

other reports in this test program no changes have 

been made.

Peer Reviewer – Goldsworthy

This study was properly conducted and 

scientifically sound. No protocol deviations were 

noted. The positive controls fulfilled the 

requirements for a valid test. The report correctly 

concludes that Gasoline ETBE Vapor Condensate 

No comments
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Comments Action/Response Page #

was negative for the induction of SCEs in rat 

peripheral lymphocytes.

Comments include:

(1) Study title should include “Gasoline ETBE 

Vapor Condensate”.

Comment has been noted but to be consistent with 

other reports in this test program no changes have 

been made.

(2) Suggest noting in the Summary Section that 

the positive control fulfilled the requirements for a 

valid test.

Statement has been added.

(3) In data discussion, it would be useful to 

comment on the observed decrease in mitotic 

index in top exposure group and whether it has 

any effect on assay results or interpretation.

Author has considered this difference of no effect 

and has not revised the discussion.

211 (b) Research Group Reviewer

p. 9, Materials and Methods:

Cell culture and Collection of Metaphases. On 

my copy [and apparently on QA copy as well, 

item #54, Audit 1/13/03], the last sentence 

describing colcemid addition is missing. It should 

read “At approximately 68 hours, 0.2μg/ml 

colcemid was added to each flask and incubated 

for approximately 4 hours.” Please check the 

master copy to be sure the sentence is included.

Added to report.

p. 11, Evaluation of test results, 1st paragraph: 

Revise sentence in Statistics to include the onetailed Dunnett’s test and trend test requested by 

EPA.

Added to report.

p. 12, Results, 4th paragraph: Revise sentence 

describing Dunnett’s test to reflect one-tail 

analysis and trend test. Adjust sentence in 

Summary referring to use of Dunnett’s test as 

well.

EPA revised statistics did not alter negative 

results for BG/ETBE

Added to report.

Agreed.

p. 19, Table 6: Please check mean SCEs per cell 

value for rat #5052. QA indicated it should be 

22.2, not 21.7 [item 57, Audit dated 1/13/03].

Report corrected.
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Sister Chromatid Exchange Satellite Report

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p. 20, Table 7: Please check AGT per group 

value for Air Control. QA indicated it should be 

26.6 (rounded to 27), not 26 [item 58, Audit dated 

1/13/03].

Report corrected.
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Immunotoxicity Satellite Report

Comments Action/Response Page #

Peer Reviewer - Schlesinger

Immunological Evaluations

Tables 1 & 2 – Is it necessary to repeat the 

methods at the bottom of each table. All this 

previously indicated information makes it hard to 

find the key to the various abbreviations used in 

the table.

While these footnotes are unnecessary in the 

context of the report, Dr White prefers to include 

them because they allow the tables to stand alone.

Peer Reviewer – Goldsworthy

This study evaluated the potential of four weeks 

of exposure to Gasoline-ETBE to affect the 

humoral immune component of the immune 

system when evaluated by antibody-forming cell 

response to T-dependent antigen sheep 

erythrocytes. This study was conducted properly 

and was scientifically sound. Data demonstrate 

that Gasoline-ETBE did not affect terminal body 

weights, spleen or thymus weight. GasolineETBE vapor condensate did result in a doserelated decrease in the spleen IgM antibodyforming cell response to sheep erythrocytes. 

Decreases in AFC/spleen were also seen in the 

two highest exposure levels. The positive control 

CPS resulted in the anticipated results.

The conclusion that, under the experimental 

conditions of this study, Gasoline-ETBE did 

adversely affect the functional ability of the 

humoral immune component of the immune 

system accurately reflects the study results. No 

effects were noted in any exposure group on body 

weight, spleen weight, thymus weight, or spleen 

cell number.

No comments needed

211 (b) Research Group Reviewer

Appendix B, Sponsors Exposure and Animal

Data: According to QA [items 59, 60] body wt, 

body wt gain, food consumption and resulting 

means tables have animals mixed among groups. 

I assume this did not occur in Appendix A. 

Individual Animal Sacrifice Data used by Dr. 

White in the report. Please resolve.

p.9, Table ES-1; As a stand-alone table, 

presentation of maximum effect at 10000mg/m3 

could be misinterpreted as indicating there was 

Issue rectified by HLS IT and tables re-run.

Footnote added as requested.
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Immunotoxicity Satellite Report

Comments Action/Response Page #

not significant effect at 20000mg/m3. Consider 

adding a footnote containing the statement that 

20000mg/m3is statistically significant data even 

though it is slightly lower than 10000mg/m3.

p. 11, Experimental Design, 1st paragraph, line 

10: Handling of cyclophosphamide is described 

twice. The sentence beginning 

“Cyclophosphamide was dissolved....” can 

probably be deleted.

Deleted.

p.12, Suggest that the last paragraph that describes 

the assay very clearly be moved to the 

Introduction (p. 10). It would fit very nicely as 

the 2nd paragraph there.

Paragraph has been moved.

Please provide a rationale for why only females 

were used in this assay. Peer reviewers have 

requested this clarification in other 

immunotoxicity studies in this series.

The immunotoxicology study is the only assay 

that used only female rats. The WG accepted the 

recommendation of Dr. Kimber White that 

females were the appropriate sex for this assay 

since in general they provide a more robust 

immune response than males.

p.15, In earlier studies, EPA inquired whether 

viabilities of 84% were considered low or 

correlated favorably with historical values. Could 

historical data from Dr. White’s laboratory be 

provided to address this inquiry? Although not 

requested in the BG/ETBE review, adding this 

information would make the report consistent 

with the others in the series.

A paragraph discussing viability has been inserted 

on page 15.

p. 16. Functional results: Dr. White indicated that 

the decrease in the IgM-antibody-forming cell 

response was dose-related. Although effects were 

induced by two doses of BG/ETBA, the response 

was not technically dose-related because the 

higher response was seen at the lower dose rather 

than at the highest dose. Please delete doserelated or revise the sentence. [also cited in EPA 

review, Aug 26, 2003, and QA item 64 Audit 

1/13/05].

Dose-related was deleted from the discussion.
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GFAP Satellite Report

Comments Action/Response Page #

EPA 

GFAP

We have reviewed the API report on the 

subchronic study for Gas+ETBE vapor 

condensate effects on GFAP submitted by Dr. 

O’Callaghan. We agree with the view of Dr. 

Callaghan that the statistically significant results 

in male rats are small and not dose-related (and in 

fact show a decrease in GFAP). The significance 

of a decrease in GFAP of that magnitude in regard 

to neurotoxic damage is unclear and probably 

represents technical variations in the assay. 

Likewise, the small, non-dose related increase in 

GFA in the female striatum and the rest of brain is 

unlikely to represent neurotoxic damage. We 

noted larger increases in GFAP levels in the 

hypothalamus (especially female) that were not 

significant. Careful evaluation of the raw data 

indicated one data point of five that was unusually 

high, and we would suggest that this is a 

dissection artifact. Overall, we agree with the 

peer review comments and the API response

No comment needed.

Peer Reviewer - Schlesinger

p. 17. Statistics. Which post hoc test was used in 

this analysis and was there any test for 

homogeneity of variance?

The statistical methods used in this assay were 

analysis of variance followed by post hoc 

comparison of treatment means (JMP®, SAS) with 

significance established at p<0.05. (p. 17 of 

report).

In addition, the notations in the text of number of 

male and female groups with changes seem to be 

in error. Two male groups rather than three 

showed effects, while one female rather than two 

showed effects. I think that the author confused 

number of exposure groups with number of tissue 

groups.

Both the reviewer and the investigator are correct. 

In general, all subsets of data within a dose group 

are considered part of that group; thus, 

statistically significant decreases in GFAP were 

seen in the cortex region and the rest of brain

sample in low dose male animals (Group II), and 

in the cortex in high dose males (Group IV). In 

low dose (Group II) females, increases in GFAP 

were observed in the striatum and in the rest of 

brain sample. However, Dr. O’Callaghan 

characterizes each brain region at each exposure 

level as a group, increasing the total number of 

groups considered.

Peer Reviewer – Goldsworthy

These studies appear to be properly conducted. 

Editorial comments from previous reviews The pituitary data have been excluded from 
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GFAP Satellite Report

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regarding the format, i.e. lack of study specifics, 

remain. Pituitary GFAP level could only be 

detected in one male one female rat; due to the 

paucity of data and inability to make any 

conclusions from this region of the brain, this data 

should be omitted from the tables and noted in the 

text that analysis of pituitary could not be 

adequately performed.

Tables 5 and 6 although the data from 1 male and 

1 female are included in Table 1, which lists 

individual animals. Text has been revised 

appropriately.

211 (b) Research Group Reviewer

p.8, Introduction: first full sentence at top of page 

– Change text as follows “Inhalation exposures to 

gasoline and gasoline plus each of the 6 fuel 

additives have been performed (replaces ‘will be 

undertaken”). Also, correct “with a two 

generation reproduction toxicity study that 

includes a neurotoxicity component” by adding 

‘for gasoline and gasoline plus MTBE vapor 

condensates only.”

Report corrected.

Although it has been decided by the Research 

group to maintain the GFAP reports in the current 

format, some general suggestions to make the 

report more consistent with other satellite studies 

are presented.

No comment needed.

p. 14-16, V. Commentary A. Background 

information: This section belongs in the 

Introduction and can be integrated with the second 

paragraph there beginning “ A universal cellular 

reaction......”

Report format has been accepted as is.

p.17, D. Typical Results: Figure 2 displays

“typical GFAP assay values from different 

regions of rat brain.” I assume these values are 

historical data from untreated control rats in Dr. 

O’Callaghan’s laboratory. Could the number of 

rats evaluated, strain, sex and age from which 

these data were collected be provided, perhaps in 

a footnote? Do these variables affect the regional 

differences in GFAP values within a species? 

Similar comments also made by Peer reviewers 

and QA.

Changed first sentence to: “Typical GFAP assay 

values obtained at this laboratory for different 

regions of the rat brain are presented in Fig. 2 (i.e. 

historical data).” And a citation for these results 

is now mentioned in the Figure Legend (p. 21) 

and reference section (Martin and O’Callaghan, 

1995).

Results and Conclusions: 
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GFAP Satellite Report

Comments Action/Response Page #

In the 3rd sentence, the Figure designation for 

levels of GFAP across different brain regions is 

incorrect. It should be Fig. 2, not Fig. 1.

Figure designation has been corrected

Delete sentence concerning “ A recently 

developed fluorescence based GFAP ELISA assay 

for pituitary data....”. Although all of this 

information is of interest, it is appropriate in the 

discussion section of a publication, not in a study 

report for regulatory submission. These changes 

have already been made in the 2nd draft of the 

gasoline GFAP report.

Section has been deleted.

Pituitary data: I am still not sure whether the 

pituitary data belongs in any of the oxygenate 

studies. Data is sketchy at best and the assay may 

not detect anything of value in this organ. 

Table 7: There are virtually no detectable GFAP 

data in any dose group except for 1 male and 1 

female at the 20000mg/m3 level. The individual 

values for these animals are much higher than 

seen in any other BG/oxygenate study where 

pituitary numbers, if present, are 0.02-0.08, not 

8.08 or 6.4. Please check these values against raw 

data [QA says these numbers are incorrect, item 

#74, Audit 1/13/03]. These data should not be 

included in Tables5 & 6, which summarize mean 

values. An average value cannot be obtained 

from only one data point and there are no pituitary 

control values. [Pituitary data issues are also cited 

by Peer reviewers and QA]. 

The pituitary data have been excluded from 

Tables 5 and 6 although the data from 1 male and 

1 female are included in Table 7, which lists 

individual animals. Text has been revised 

appropriately.