Inhalation Toxicity Report

Comments	Action/Response	Page #

Gasoline Ethanol Vapor Condensate:  A 13-week Whole-Body Inhalation
Toxicity and Neurotoxicity Assessments including GFAP, and 4-week In
Vivo Genotoxicity, and Immunotoxicity Assessments (by Huntingdon Life
Sciences, Study No. 00-6127, Sponsor No. 211-EtOH-S)

EPA

ORD finds that, in general, the main studies and the specific
neurotoxicity, genotoxicity, and immunotoxicity assessments included in
the reports were conducted according to EPA test guidelines.  At this
time, however, we cannot recommend approval of the study reports as they
are, because we have identified significant deficiencies in the conduct
of the studies and the statistical analyses and reporting of data in
some of the assessments.  Similar issues were described previously in
our memorandum to you of May 21, 2002, regarding the subchronic toxicity
study for baseline gasoline.  The attached memoranda provide specific
comments on each of the study reports. 	This was the original draft
report of these BG+EtOH 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

No additional comment needed

	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).  Additionally, the ORD team reviewed the TSCA 8(e)
notification associated with the immunotoxicity and GFAP findings from
the subchronic study on gasoline ethanol vapor condensate.  Our main
concerns are discussed in the attached memoranda.

ORD strongly encourages 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. 	Comments
from EPA and other reviewers are addressed here and submitted with the
second draft report of this study.

No additional comment needed

	While in general the NHEERL reviewers concluded that the studies were
conducted in accordance with the testing guidelines and that the results
supported the stated conclusions, there were also significant exceptions
involving the conducts 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 previously noted, we have the following
assessment of the gasoline plus EtOH reports.

	No comment

	Micronucleus Assay and Sister Chromatid Exchange Satellite Study

The micronucleus assay and the Sister Chromatid Exchange Satellite Study
(SCE) assay appear to have been conducted according to recognized and
appropriate protocols.  The conclusions of no effect of gas + EtOH are
supported by the data.  The reviewers’ minor comments have been
adequately addressed.	No comment

	Immunotoxicity Satellite Report.  These studies were conducted to
determine whether 4 weeks of 5d/wk exposure (0, 2000, 10,000 or 20,000
mg. condensate/m3) to a mixture of ethanol and gasoline vapor condensate
affected lymphoid organ weights (spleen and thymus), spleen cellularity
or the T-dependent antibody response to sheep erythrocytes.  A separate
group of animals, not subjected to exposure chamber conditions, was
given daily injections of 50 mg cyclophosphamide/kg of body weight over
the last 4 days of the exposure period.  Cyclophosphamide (CY) is known
immunosuppressive drug and was included as a positive control.  Rats
were immunized prior to sacrifice by intravenous injection of sheet
erythrocytes.  The methods employed were appropriate, results are
clearly described and the conclusions are supported by the data.	Summary
of study design.  No comment	Study located in Appendix Z

According to OTAQ (John Brophy), the condensate was 10% EtOH by volume.
However, I could not find this information in the reports that I
downloaded from the API site.  It would be helpful to include that
information in the Executive Summary and Experimental Design section of
all discipline-specific reports.	This information is available in
Appendix S in volume 4 of the 13-week Subchronic study.

Since the Satellite studies are now appended to the subchronic study no
addition sample description is considered necessary within those
subcontractor reports.  In addition, a separate document providing the
condensate data for all test samples will be prepared and submitted to
EPA.

An entry has been added to Section 2.4.3 (p.18) of the subchronic report
referencing Appendix S.  Done 	p.18 Subchronic report

Terminal body weights of the positive control group (CY) are not
mentioned in the text of RESULTS, although they are included in Table 1.
 All other variables described in the text include data for the CY
group. 	A sentence has been added to the results indicating that CY
treated rats showed a statistically significant 8% lower body weight
compared to controls. Done

	Appendix Z page 13

The 2nd RESULTS paragraph states that condensate exposure had no effect
on relative or absolute spleen weights, but Table 1 seems to imply a
significant (p=0.05) trend for relative spleen weight change.  It
isn’t clear whether values for CY-exposed animals were included in the
trend analysis.  If they were, why were they?  If they were not, then
either the text or Table 1 seems to be in error. 	CY-treated animals
were not included.  Both the text and table are correct as written.
There is no statistically significant difference in weight, but there is
a trend, which is statistically significant by the Jonckheere's Test.
However, due to the liberal nature of the Jonckheere's Test limited
biological emphasis is placed on this statistical finding.

No change –OK – Note: This decision was Dr Kimber White’s	Appendix
Z. page 14

The viability of spleen cells is reported to be greater than 84% in all
groups.  This seems to be a bit on the low side.  Is this within the
historical range established by ImmunoTox, Inc.?  How variable were the
data?  I assume that no treatment-related effects on viability were
found, but that is not stated in the text.

	The results are within historical range.  Viability of spleen cells
does not correlate with functional activity evaluated and is only used
as a gross parameter of the general well being of the splenic
population.  Most of the decrease in cell viabilities is related to cell
types not being evaluated.

No change –OK – Note: This decision was Dr. Kimber White’s
Appendix Z. page 15

In Conclusions, the report states that the observed decrease in antibody
producing cells was “not unexpected, based on the known immunotoxicity
of ethanol,”  Perhaps it is beyond the intended scope of the report,
but to fully appreciate the immunotoxicology data, the levels of
exposure in the present study should be put in context by comparison to
the published ethanol immunotoxicology data, even if the comparison is
made in a sentence or two.  For example, Steve Pruett’s work is mainly
based on acute EtOH doses that produce blood alcohol levels in the range
of 0.4-0.5% in mice, and feeding studies in mice and rats typically use
EtOH concentrations that deliver 20-30% o more of the animal’s daily
caloric intake as ethanol.  If the concentration of EtOH in this study
was indeed 10% (see comment #1), then rats were exposed to 0, 200, 1000
and 2000 mg/m3 EtOH.  Were blood alcohol levels measure at the 3 vapor
concentrations?  If those data are not available in the API report (and
they should be, given the wealth of animal and human data we have at
various BAL), the report should include an estimated daily intake of
EtOH, based on EtOH concentration, rat respiratory rate, duration of
exposure, etc.  Using the known or estimated intake values, one could
then state whether the antibody response in these studies was suppressed
at EtOH levels comparable to those in the published literature.  For
example, results could be compared to Bagasra et al. (Immunology 61,
63-69, 1987 who reported enhanced T-independent antibody responses, and
initial elevation followed by suppression of the T-dependent response to
sheep erythrocytes responses in male* Sprague-Dawley rats consuming 36%
of calories as EtOH.  If the actual or estimated daily intake of EtOH
was considerably less than levels reported to suppress the antibody
response, would that suggest synergistic effects of gasoline vapor and
EtOH?  As note above, this discussion may be beyond the scope called for
in this process.  If that is the case, maybe these comments will be
helpful if and when the data are developed into a manuscript.	Dr. White
did not make any additional comments in the report but these
recommendations will be included in any subsequent publication.

Blood alcohol levels were not measured for this study.

	*Why were only female rats evaluated?  At least one report (Grossman et
al., Alcohol Clin. Exper. Res. 17, 832-40, 1993), reported a degree of
gender sensitivity of S-D rats to EtOH, with males more sensitive than
females in some of the measured endpoints. 	The immunotoxicology study
is the only assay that used only female rats.  The WG accepted the
recommendation of Dr. Kimber White, study director, that females were
the appropriate sex for this assay.

	FOB and Motor Activity in Suchronic toxicity study.  This protocol has
been reviewed previously (Baseline Gasoline study), and many of the same
issues apply here as well as the other gasoline studies.  After the
previous 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, I will only summarize
those issues. 	No comment

	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.  This issue was also raised by Dr.
Schlesinger in his review.  We have been in discussions with the
sponsor’s statistician, who 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 then analyzed.  Gender should be included as a factor in the overall
ANOVAs for all endpoints. 	The revised statistical evaluations have been
completed and are included in the second draft report of this study  OK

	pp.45-46 [Methods]

pp. 54-55 [Results]T

Appendices G, H

Table 7

We have concern regarding the landing foot splay data.  The data from
the current study underscore the need for analyzing the means of the
trials, not the individual trials.  Landing foot splay was increased in
female rats at 13 weeks, but only one of the two trials – this finding
is uninterpretable.  The control data seem to decrease by a third over
the 13 week study, which could account for the apparent increase in all
treated groups.  However, we withhold judgment on these data until the
statistical analysis is corrected.  We understand that the revised
analysis is underway and will be submitted when available.	The revised
statistical evaluations have been completed and are included in the
second draft report of this study OK

	p. 54; Table 7

Neuropathology and GFAP.  We have examined the neuropathology sections
of the study reports, and based on the information provided, we conclude
the neuropathological assessment was conducted in accordance with the
EPA Neurotoxicity Screening Battery Guidelines, and the data are
consistent with the conclusion of the report that there is no evidence
of treatment-related neuropathology. 	No comment	Appendix AA

We also reviewed the study reports with regard to effects on glial
fibrillary acidic protein (GFAP).  There is good agreement in the levels
of GFAP in control animals between the baseline study and the gas + EtOH
study just submitted.  We concur with the conclusions in the report that
there are significant effects on GFAP in male rats.  The small changes
and lack of dose-response of raise questions concerning the biological
significance of these effects.

	No comment

	Since the GFAP analysis is potentially informative with regard to
neuropathology, the GFAP findings were examined in relation to the
neuropathology section of the report.  As noted by one of the previous
peer reviewers, the statement in the summary that the
treatment.”…did not elevate the levels of GFAP in any of the nine
brain regions examined” is incorrect and inconsistent with the
discussion of the results in the report.  Significant increases in the
level of GFAP were observed in 6 of 8 brain regions (excluding the
pituitary).  These increases did not appear to be dose-related and were
observed in all dose groups for one brain region, the cerebellum.  The
elevations were in the range of 25-50% above control values.  The
variability in control values compared across companion studies for
certain brain regions appears to exhibit a similar range. 	The EPA
reviewer is correct.  The text in the Summary was a text error and did
not reflect the actual results of this study.  The section has been
rewritten to accurately present the findings of this study in the second
draft.  OK – Entry in Summary table on p. 9 is also correct.

Suggested Summary Table attached.

Summary table replaced.	AppendixAA

page 7

Subchronic report p. 9

Based on concerns regarding the appropriate interpretation of the
increase GFAP levels in the cerebellum of all those groups relative to
controls, we performed two statistical analyses on the available data:
1) comparison of control values between the studies and, 2) an analysis
of the dose-dependence of GFAP levels in cerebellum and striatum in the
EtOH study.  This preliminary analysis indicated that the effects on
GFAP in cerebellum are significant at all dose levels and the
differences in control values between studies are not likely to account
for these effects.  While the biological significance of these small
changes in GFAP levels is unknown, the discussion of the results in the
report indicates treatment-related increases in GFAP may be indicative
of injury.  The summary of the report, however, does not address these
observations and concludes that 10,000 mg/m3 is a NOAEL.  If the
observed effects on GFAP levels in the cerebellum are considered an
adverse effect, then 2,000 mg/m3 would be considered a LOAEL.  Given
such potential implications, we recommend two steps be taken to assist
in ascertaining the significance of the GFAP alterations in the
cerebellum.  First, as previously noted by other peer reviewers, the
procedures used for statistical analysis of the GFAP data need to be
clearly articulated.  Second, further neuropathological examination of
the cerebellum may provide a more compelling basis to ascertain whether
or not the increased GFAP level reflect injury.  Such an examination
would include a comparison of control animals with animals from all dose
groups, sections from at least three levels through the cerebellum and
the use of special stains.

The summary has been rewritten to accurately reflect the data.  OK

It has been determined by the RG that a summary table listing results of
all studies with NOAEL and LOAEL for each study be included in the
Subchronic toxicity report.  Overall NOAEL and LOAEL values for the
entire study set will not be determined.

Entry for Immunotoxicity study in Summary table on p. 9 is incorrect and
will be corrected for the final draft 2 review by the contractor.  The
GFAP entry is correct on this table.

Summary table revised

No additional neuropathologic examinations were performed. OK

Appendix AA p. 7

Page 9, subchronic report

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 the baseline gasoline
vapor condensate study recur in the current report.  To these general
comments, we add a request to better specify the composition of the
tested substances especially in regard to the proportion of the additive
in the gasoline.  We understand that these general issues will be
addressed in a revised report.  In addition to the issues raised here
specific to the gasoline plus EOH 13-week study.  Among the issues to be
addressed specifically for the gas plus EtOH study are points regarding
the reporting and interpretation of the immunotoxicity and neurotoxicity
data as described above.  	No comment.  General issues cited here are
addressed elsewhere in this checklist and in the second draft of this
report.

	Peer Reviewer – Dr.Schlesinger

Main Study:  All section and/or page numbers and comments in the Main
Study section below refer to material from Volume I of the Main Study
Report.

There are a number of places where it is stated that the exposures were
“generally” 5 d/wk, etc.  If there were instances where they were
less than this, then the total number of actual exposures for each group
should be stated.  Or, perhaps the range of excursions from this
generality could be provided	Animals were exposed to BG + ETOH VC for a
minimum of 65 exposures over 13 weeks in the Main subchronic study, for
neuropathology and for GFAP (see pp15-16).  Clarified in the Summary and
M&M  “for at least 65 exposures”. OK

	Pages 5 and 16 and 25

What was the reason that clinical laboratory studies were not performed
with the Recovery animals?  This may have perhaps shown some delayed
effects. 	It was decided by the WG at the outset of the program that
hematology, clinical chemistry, opthalmology and neurobehavioral tests
would not be performed on recovery group animals if no significant
effects were observed in animals during the exposure period.  Past
testing on gasoline, gasoline blending streams, and neat oxygenates had
not demonstrated delayed expression of toxicity after exposure was
terminated that had not been seen in exposed animals.  In the BG + ETOH
VC study the changes observed in clinical pathology parameters were not
considered to be of sufficient statistical or biological significance to
warrant testing of recovery rats.

	Methods.  Neurobehavioral Studies.  It is stated that testing was done
on nonexposure days.  Was testing done on weekends or were animals
undergoing these tests not exposed for the full 5 d/w when these tests
were to be performed. 	For neurobehavioral testing, rats were not
exposed on behavioral test days (Sect 2.13, p. 29 of report).  However,
these exposures were made up later (either on week-ends or with
additional exposure days prior to necropsy), to provide consistent
number of exposures for all rats.  Checked OK	p. 30

Methods: Statistical Analysis.  It is stated that evaluation of the
means was made by the appropriate test.  What was the criteria for
appropriate? 	See response below

	When the Bartlett’s test indicated nonhomogeneity of variance, the
investigators then went on to use a nonparametric analysis of variance.
An alternative approach would be to use some transformation, which 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. 	A single
computerized statistical testing package was employed for all parameters
listed on p.42-43 of report.  In this package, analyses by individual
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.

Agreed – text appears the same in all reports.

	pp. 44-45

It is also stated that statistical tests were conducted in a number of
cases at both the 5% and 1% significance levels.  The justification for
this is not apparent.  One of the a priori decisions which is to be made
before the study is conducted involves choice of the appropriate level
of significance.  Only one should be selected based upon whatever
criteria the study director chooses and only this level should be
reported.  There needs to be consistency throughout the entire study.
These levels of significance are pre-selected since they are inherent in
the laboratory’s computer statistical package.  In practice,
Huntington Life Sciences employs the 5% level to indicate statistical
significance Whenever data are significant at the 5% level it is
reported as statistically significant in reports.

Agreed – text appears the same in all reports.	p. 45

The description in this section is not clear on all aspects of the
statistical analysis.  It is presumed from the manner in which the data
area subsequently presented in the report that the males and females
were separately analyzed using the techniques described.  Is there any
reason why the genders were not analyzed together for each endpoint
using a two-way ANOVA?  While it is stated that analyses were performed
for each gender separately and then combined, this does not make it
clear whether the genders were evaluated separately in one analysis or
whether data were evaluated using one way ANOVA by combining both males
and females into one group.  In any case, what was the justification for
the analyses being performed both separately and combined?  Were there
some criteria for evaluation of the results obtained with each of these
separate assessments.  	See comments above concerning laboratory-wide
use of computerized standard statistical package.

	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 comments
above.

	Was any statistical evaluation of the chamber atmospheric data
performed to assess consistency?  	No statistical evaluations were
performed.  Consistency of chamber atmosphere was verified only
analytically during the exposure series.  OK

	Section 3.1 Chamber Monitoring (p. 46).

It was found that there were differences between the nominal and
analytical exposure concentrations that were ascribed as most likely
being due to “…slight inaccuracies in the calibration of the chamber
airflows and the calibration of the IR monitor”.  This rationale is
not quite clear.  The nominal exposures were obtained using the airflow
parameters, so this would clearly be affected by any offset of airflow
calibration.  However, the analytical levels were obtained in real time
with the IR monitor at various times during the study.  Thus, this
rationale assumes that the monitor was not calibrated properly
throughout the entire study, and this should have been obvious well
before the study ended and the exposure data were evaluated after the
fact.  In any case, the nominal and analytical mean values differed by
16-43%, depending upon the exposure group.  This is not a trivial
difference and it begs the question as to which conctration is the
“real” exposure level?  Note- Dr Goldsworthy also cited these
differences.	This variability was identified during the pre-study
chamber trial, continuing throughout the study.  A variety of approaches
were employed to resolve the differences.  These included, checking
pressure differentials and recalibrating air flow monitoring equipment,
re-checking canister weight determinations, and applying alternative
programs (linear plotting vs curvilinear, polynomial based plotting) to
determine nominal concentrations. At this study’s initiation, a Miran
calibrated at a wavelength (the wavelength employed for the Baseline
gasoline study and the BG+MTBE study) was used in parallel with the
Miran calibrated specifically at a wavelength for BG+ETOH.  This data
demonstrated the Miran measured values to be up to 14% greater than the
nominal values for the former Miran but up to 50% less than the nominal
values for the latter Miran.  A decision was made at that point to
proceed with the latter Miran since the nominal value should be greater
than the measured value.  Subsequent to this study’s completion and
prior to the 1-generation study’s initiation, additional evaluations
of the Miran were conducted.  These evaluations indicated that the Miran
calibrated for this study was calibrated at a wavelength that was highly
sensitive to the ethanol concentration and less sensitive to the overall
total hydrocarbons concentration.  It has been speculated that ethanol,
being more water soluble than the hydrocarbons in the vapor condensate,
may have solubilized and remained behind in the chamber.  Therefore, a
Miran calibrated to be overly sensitive to ethanol may have given a
falsely low chamber concentration if some of the airborne ethanol was
solubilized and remained behind in the chamber.  Such wide differences
between nominal and analytical values were not seen with any other
BG+oxygenate sample tested in the 211(b) program, indicating that
ethanol had a significant impact on atmosphere monitoring methods.
However, in all instances, the analytical concentration was equal to
target concentrations while nominal values were in excess of target
concentrations, demonstrating that animals were exposed to levels of
gasoline+ethanol vapor condensate well above likely human exposure and
high enough to effectively evaluate potential toxicity.  The analytical
values are considered definitive for this study.  In contrast, the
subsequent 1-generation study used a Miran calibrated at a wavelength
(the wavelength employed for the Baseline gasoline study and the BG+MTBE
study) that was less sensitive to the ethanol concentration and more
sensitive to the overall total hydrocarbons concentration.  This
study’s Miran measured values were within 7% overall of the nominal
measured values.

More descriptive text has been added to R&D and Summary.

The “More descriptive text …” in the 2nd draft should be rewritten
to exclude the discussion of “solubilized” ethanol in both the R&D
and Summary sections (pp. 51-52; Appendix S)  All oxygenates in the 211b
testing program are more water soluble than the hydrocarbons in gasoline
yet there was no consistent problem with measuring test atmospheres.
There is no reason to speculate on the concept of “solubilized”
ethanol when the BG+ETOH 1-generation reproduction study demonstrated
that a properly calabrated Miran instrument gave measured values within
7% of nominal values.  The study text should stick with the facts that
nominal values were significantly higher than measured values in the
subchronic study demonstrating that animals were exposed to levels of
BG+ETOH vapor condensate high enough to effectively evaluate potential
toxicity.  Instrument calibration was improved in later studies with
BG+ETOH.

Text Revised.	Pages 6 and 49-50

In comparison of neat and test atmospheres, it was noted that there was
a difference in ethanol levels, with those in the latter being
consistently less than those in the former.  The investigators partially
attributed this to a problem with the extraction method used.  The first
question is “why was not the problem found earlier than after the
study ended?”  In other words, was there not a test of the neat
substance and a mock generated atmosphere to allow comparison before the
exposure actually began.  Furthermore, if the extraction problem was
only part of the rationale for the discrepancy, what was the basis for
the rest of the problem?  Finally, in the Analytical Report, Appendix S,
it is noted that the ethanol levels were corrected for reporting
purposes using the extraction efficiency obtained from an experiment in
which both carbon disulfide and butanol were employed during extraction
of spiked samples.

Perhaps, the better approach would have been to use spiked samples and
the original method, i.e. without the butanol.  This would have provided
the actual extraction efficiency used throughout the study.

Note- Dr Goldsworthy also cited the variability in EtOH levels.	The
analytical differences were identified during the course of the study
and alternative collection and extraction procedures were tested to
develop an appropriate ethanol correction factor.  The issue was not
fully identified until comparisons were made with the GLP pretest
analysis of the neat material performed at another laboratory and
received after the study was underway.  Carbon disulfide extraction is
most effective in collecting hydrocarbon and oxygenate fractions from
carbon sampling tubes for GC analysis but was not as effective for
ethanol resulting in ethanol values approx. 20% below expected.  Butanol
is a better extractant for ethanol, producing close to 100% extraction
efficiency, but a poor extractant for hydrocarbons.  Neither alone is
adequate to deliver accurate distribution of all components from the
BG+ETOH chamber atmosphere.  Therefore, the ethanol correction factor
was developed using butanol extraction, and applied uniformly to all
weekly sampling data after the study was completed to provide an
accurate profile of component distribution.    OK

In fact, a spiking procedure was employed in real time.  Each weekly
analytical run included 2 test material standards (TM-1, TM-2) and two
spiked controls.  The spiked controls and chamber samples gave similar
ethanol profiles while the TM standards were approximately 20% higher.
However, in order to most accurately evaluate the proper correction
factor, a separate experiment was performed after the study and the
butanol-based ethanol correction was applied after the biophase was
completed.  These data are available in Appendix S of the study.  OK

Dr. Goldworthy seemed satisfied with the analytical correction employed.

Overall, comparison of analytical characterization over time indicated
that the ratios of hydrocarbons in the chamber were comparable to the
neat test substance and the correction for ethanol extraction brought
that value into appropriate relationship. 	pp 51-52

Appendix S

The air control chambers contained particles.  What were these and why
were there not appropriate filters on the chamber intake to remove
particles from the incoming air?  While the data support the conclusion
that the atmospheres were in vapor form and not in particulate form, has
any thought been given to the possibility that these “background”
particles which were also present in the vapor atmospheres could have
acted as modalities for the condensation of the vapor and a vector for
delivery of the vapor to areas it may not have reached if inhaled in the
absence of such particles?  For example, if the vapor deposited in the
respiratory tract at a more proximal level due to its adsorption onto
some of the particles while the pure vapor may have reached more distal
sites, then the ultimate results of the study would reflect this
difference in dosimetry.  In the rat, particles of the size measured
would have significant deposition in the upper respiratory tract and
proximal lung airways.  The investigators need to address this potential
issue. 	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.  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 addition, no unique particle
disposition was observed microscopically in lungs or nasal passages.  OK

	p. 51

Section 3.4 Body Weights (p. 49).  It is noted herein that the male
weights were only statistically significantly different from controls
during the first three weeks of exposure.  However, from Figure 2, the
weights appear to be consistently different throughout the study and
with the differences being relatively the same as during the initial
weeks of exposure.  Furthermore, differences in females are indicated as
being “slight”.  This is a subjective term; the differences seem to
be around 10% and I am not sure this qualifies as slight.  It is also
noted that “the differences in weight between the Air Control animals
and the 20,000 mg/m3 exposed animals abated during the 4 week recovery
period.”  In examining Figure 3, this difference seems to clearly
remain in females during the recovery period, and it also seems to
remain in males as well (Figure 2). 	The RG agrees that body weight
effects should be more fully discussed in the text and will develop
appropriate additions with the study director, similar to the discussion
below.

Male rats exposed to 20,000mg/m3 gained significantly less weight than
any other dose group compared to negative controls between wk 0 and 3 of
exposure.  Thereafter, weekly wt gains were comparable to controls but
were never sufficiently greater than controls to make up the difference
from the early weeks of exposure.  These weights were not statistically
significantly lower than controls.  This pattern suggests adaptation to
exposure conditions and return to normal weight gain.  The randomly
assigned recovery high dose males similarly began the recovery period
with lower average wt and wt gain than controls but showed comparable wt
gain during the recovery period, probably correlated with increased food
consumption, not sufficient to make up the weight differential with
controls but not statistically significantly below control.  A similar
trend was observed in high dose females with more instances of
statistically significant lower body wt and body wt gain from weeks
2-13.  The observed lower body wts and wt gain for both sexes in the
high dose group, approximately 10% in females, are indicators that a
maximum tolerated dose had been reached in this study.  Text has been
added. OK	Pages 6-7 and 53

Section 3.6.  Feed Consumption (p. 49).  It looks like (Figure 4 as if
there was an increase in feed consumption in males after the exposures
to the highest concentration ended, and which returned to normal by the
end of the Recovery period.  While this may not be of biological
significance, this pattern should be note.  Furthermore, while the
investigators note that the effects did not show a “treatment related
pattern” they clearly only occurred at the highest exposure level.
The fact that they did not occur at the mid and lower exposure levels
does not mean that they are not “real,” as implied in the text.
This reviewer has found with a number of inhaled chemicals that effects
may occur at one level and not at a higher one, for a number of reasons.
	High dose females also increased their food consumption after exposure
ended and continued to eat more than controls throughout the recovery
period except for wk 16.  It should not be construed that increased food
consumption is not considered a “real effect”, only that it is not
considered adverse.  These observations contribute to the likelihood
that 20,000mg/m3 was a maximum tolerated dose, and that animals
responded with increased appetite after exposure was terminated.  The
information for both sexes in the control and high dose recovery groups
has been added to the text and sentences rephrased as appropriate.  OK

	Pages 7 and 53-54

Section 3.7.1.  Motor Activity (p. 49).  The discussion in Table 7 of
the results of the statistical evaluation of the motor activity data is
somewhat confusing.  Perhaps it could be simplified so that the reader
knows what is and what is not of significance.	Revisions of this section
have been made to improve clarity.  Alternative statistics recommended
by EPA have been performed on motor activity data and selected FOB tests
and will be incorporated.  OK

	Page 54

Section 3.7.2.  FOB (p. 50).  The data in Table 8 show that there was an
increase in landing foot splay that appeared to be relatively consistent
across all of the exposure levels and was significantly different from
control.  As noted in a previous comment findings that effects are not
directly correlated with exposure level do not mean that they should be
discounted.  Perhaps some effects are “all or none” and could be of
toxicological significance.  Furthermore, the use of dual levels of
statistical significance should be avoided. 	Review of wk 13 trial 1
data indicate that the statistically significant increases were similar
across all dose groups and that the negative control values was
substantially lower in trial 1 (3.4) than in trial 2 (4.0), which was
probably responsible for the statistical significance.  Each trial was
analyzed separately and trial 2 showed no significant differences at any
dose level.  It is certainly appropriate to cite this finding in the
text but its biological significance may be limited.  Trials 1 and 2
have been averaged as per prior EPA advice and averages evaluated.  The
differences seen in landing footsplay addressed by Dr. Schlesinger were
not mentioned specifically in the report results text because the
statistical analysis report using EPA recommended statistics (Table 7)
did not consider them as significant.	Pages 54-55

Section 3.8.1; 3.8.2.  Hematology; Coagulation (p. 50).  There appear to
be three criteria for concluding that effects are not biologically
significant, as noted in these and some other sections of the Report.
One is the lack of a dose-response relationship, as noted above; another
is whether values were comparable to historical controls; and a third is
whether effects at 4 wk are still seen at the terminal sacrifice time.
A suggested reason to avoid the first criterion is provided above.  The
second criterion should not be used, as it is more appropriate to
compare effects only to concurrent controls, since some “unknown”
factor or factors could have affected controls in this study only...
Finally, the third criterion is inappropriate, as effects could occur
early on during a study, and then resolve.  This adaptation phenomenon
is seen

with some other inhaled chemicals and does not mean that the chemical
has no significant adverse effect.

	Both Drs. Goldsworthy and Schlesinger requested more detailed
discussion of statistically significant changes observed in hematology
and clinical chemistry parameters.  They differ in opinion concerning
the extent to which comparison with historical data, and the consistency
of occurrence over both sampling times (wk 4 and 13) should be used to
determine biological significance.  Dr. Goldsworthy tends to agree with
the report that statistically significant changes which fall within the
historical range and/or occur only at wk 4, resolved by 13, are not
likely to be biologically significant.  Dr. Schlesinger sees limited
value in comparison with historical data, and is of the opinion that
effects seen at one sampling interval and not the later one does not
mean that the chemical has no significant adverse effect.  He is also
concerned about discounting as not biologically significant, effects
which occur only at lower doses in the absence of a dose-response.
However the biological significance of variable statistically
significant changes in clinical pathology parameters in subchronic
studies is difficult to determine in the absence of overt physical
effects or histologically observed tissue changes.  The study duration
is comparatively short and individual animal variability may produce
wide standard deviations.  Comparison with historical control ranges are
valuable in sorting out these data, just as the use of ranges of
acceptable values for blood parameters in human analyses is valuable.
Changes which occur at low doses, especially in only one sex, but are
not seen in higher doses in a dose-related pattern are usually
discounted in the absence of corroborating pathology, especially if
effects are seen only early in the study and not after continued
exposure.  As Dr. Schlesinger pointed out, some statistically
significant values appear to be little different from the controls, and
it would be inappropriate to attach biological significance to data
points which owe mathematical significance to the vagaries of
computerized statistical packages with pre set levels of sensitivity.
Text in the clinical pathology section has not been expanded to include
historical control data, although that data are available at HLS upon
request.   OK	pp 55-56

A number of values in Tables 9 and 10 are noted as significantly
different from air control, yet the means and errors are so close to
control that it is difficult to see how they can be different.  Could
this be an aberration due to the statistical test used?

	As Dr. Schlesinger pointed out, some statistically significant values
appear to be little different from the controls, and it would be
inappropriate to attach biological significance to data points which owe
mathematical significance to the vagaries of computerized statistical
packages with pre set levels of sensitivity.  The text has been revised
to address the statistical but not biological significance of these
values. OK	Pages 55-56

Section 3.8.3.  Clinical Chemistry.  (p. 51).  There seem to be a number
of consistent effects in various parameters at the 4th week interval,
both in clinical chemistry and other endpoints including kidney weights.
 Perhaps these effects are of toxicological significance with short term
exposure even if they resolve by the end of the 13th week exposure
period and should be discussed in light of this apparent pattern. 	See
comments above

Most of the wk 4 clinical chemistry effects are cited on p. 51.  In the
context of this study alone, it is difficult to assess biological
significance as more than random occurrences.  However, upon completion
of the 211(b) program, comparisons across all the 13 wk studies may
reveal patterns of response.  OK	Page 56

It is stated that some differences were “minimal.”  This is a
subjective term and perhaps some actual percentage differences could be
given so the reader can make up their own mind about this. 	Since actual
average values are presented in Table 11 and appendix K, percent
differences can be calculated by the reader.  The study director
recommended that adding this calculation to the text would not be of
value to the reader for values not judged of toxicological significance.
 No change  OK

	In Table 11, some control values for DBILI are listed as 0.  Is this a
real value or was there none detected.  If none was detected or levels
were below the detection level of the procedure, then it is questionable
as to whether the statistical testing used is appropriate.  Furthermore,
some of the absolute values obtained with exposure are so close to
control that it is difficult to see how there can be a statistically
significantly difference.	In addition to the female data sets, there are
similar 0.0 entries for all male Direct Bilirubin (DBILI) entries at wk
4 and values for gamma glutamyl transferase (GGT) were 0.0 but included
standard deviation values of 0.5-0.4.  The accuracy of these data
entries has been reviewed by the study director and are correct as
presented within the limits of the computer system rounding methods.
Where statistically significant values appear to be little different
from the controls, it would be inappropriate to attach biological
significance to data points which owe mathematical significance to the
vagaries of computerized statistical packages with pre set levels of
sensitivity.  OK	Table 12 of second draft

Section 3.9   Organ Weights (p. 53).  The footnote in Table 12 indicates
that two different statistical tests were performed on the same data.
This requires some justification.	The study director presented a
rationale for use of two statistical tests for organ weights: Organ
weight data is analyzed by parametric procedures using the Dunnett’s
test for homogeneous data and the modified T-test for non-homogeneous
data.

OK.  See p. 44 Statistics

	p. 44;

Table 13

Section 3.10.1 Macroscopic Pathology (p. 52).  There seems to be a
consistent pattern of lung discoloration in a certain percentage of
animals at all levels of exposure, but with the incidence rising at the
mid and high exposure level.  This observation is not really addressed
in the text. 	At terminal sacrifice, discoloration of the lungs was
observed in Control (1M, 2F), low dose (2M, 1F), mid dose (7M, 3F), and
high dose (5M, 4F); in recovery, lung discoloration was observed in no
control rats and 1F high dose animal.  No structural histological
correlates were observed in lungs of female rats, while lungs of a few
male rats (2 control, and 1 mid dose) had minimal inflammation.  The
study pathologist considered the presence of discolored foci in lungs in
the absence of structural histological correlates to be incidental
findings in animals exposed by inhalation.  Additional clarification has
been added to text.   OK	Pages 57-58

Peer Reviewer – Dr. Goldsworthy

The potential inhalation toxicity of G-Ethanol 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 and 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 deviation
referring to misplaced peripheral blood smears from 4-weeks evaluation
had no effect on the study results.  Review of in-life test parameters,
i.e., temperature/humidity out of range excursions, were occasionally
quite high (98% humidity, 31°temperature) but had no effect on the
overall study results as indicated by the lack of any associated in-life
or histopathological assessments. 	No comment

	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.
It is not clear why this was not noticed in the pre-study test
conditions (where chamber conditions are optimized) or further examined
after these differences were noted after the first few weeks of
exposure.  No differences were note in particulate levels and, with the
exception of ethanol, animals were consistently exposed to all major
components of the test substance in their proper proportions.  Measured
levels of ethanol in the test atmosphere was approximately 20% less than
those measured in neat substance.  This was apparently due to extraction
inefficiency.  Given this minor circumstance, and the fact that
characterization of the test substance did not occur prior to the
experimental initiation (page 2), it is worth commenting on the results
of the Sponsor’s purity analysis conducted at a later date (page 17).
In general, the exposure techniques were suitable to adequately test the
potential toxicity of G-Ethanol.  	Dr. Schlesinger also commented on the
variability of EtOH levels.  The analytical differences were identified
during the course of the study and alternative collection and extraction
procedures were tested to develop an appropriate ethanol correction
factor.  The issue was not fully identified until comparisons were made
with the GLP pretest analysis of the neat material performed at another
laboratory and received after the study was underway.  Carbon disulfide
extraction is most effective in collecting hydrocarbon and oxygenate
fractions from carbon sampling tubes for GC analysis but was not as
effective for ethanol resulting in ethanol values approx. 20% below
expected.  Butanol is a better extractant for ethanol, producing close
to 100% extraction efficiency, but a poor extractant for hydrocarbons.
Neither alone is adequate to deliver accurate distribution of all
components from the BG+ETOH chamber atmosphere.  Therefore, the ethanol
correction factor was developed using butanol extraction, and applied
uniformly to all weekly sampling data after the study was completed to
provide an accurate profile of component distribution.   OK	pp. 51-52;
Appendix S

Experimental data revealed no effect of G-Ethanol 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 differences noted in body weight gain.  This
was especially true in high-dose exposed female rats from study week
2-13.  Summary (page 7) should be reworded to change the presentation
from “during the latter 12 weeks to “weeks 2-13”.	Text in summary
has been reworded.  OK

	Page 6

Male rats at the high dose had consistently lower body weights through
the entire exposure period and recovery, but this was statistically
significant only at weeks 1-3.   For both summary (page 7) and results
section (page 3-5), it is misleading not to note this trend and to
present the data at terminal in males as “essentially comparable”.
Revision of this section of the report is cited above in response to Dr.
Schlesinger’s body weight comments. OK	Pages 6-7 and 53

Additionally, the presentation of data for the recovery animals is
stated only to be ‘abated’ when, in fact, the body weight decreases
observed at this time are quite similar (although not statistically
significant) to that observed at terminal and similar to the trend seen
in high dose exposed rats throughout the 13 week exposure period.  This
should be noted. 	Revision of this section of the report is cited above
in response to Dr. Schlesinger’s body weight comments. OK	Pages 6-7
and 53

No general or consistent findings were observed in hematology,
coagulation and clinical chemistry in exposed animals.

However, text (page 50) mis-states some of the minor differences
observed in these data sets.  Text (page 50) states that differences
were only noted in erythrocytes counts; Table 11 demonstrates
statistical differences in HgB and HCT as well (at terminal).  It is
appropriate to dismiss the few data differences observed in coagulation
unless because these values fell into the historical control range of
values (it would be useful to list the historical range) and the fact
that this effect wasn’t seen at both intervals. 	The statistically
significant decreases in Hgb, Hct and RBC in the high dose males at 13
has been addressed in the text.  Historical control values were not
added to the report but are available at HLS if needed.  OK	Page 55

Some clinical chemistry values i.e., chloride values were seen in both
male and female exposed rats and thus may be related to exposure.  The
minor differences noted and the lack of consistent responses between the
4 and 13-week intervals lead to the appropriate conclusion that these
changes are not exposure related.  However, the comment that these
values ‘generally’ fall within laboratories historical control
ranges does not provide added assurance.  Specific comments on which
values were out of range would be useful.  Text may also wish to comment
on the very large standard deviation values noted in bile acid values.
Increases in chloride (male & female), sodium (male), bile acids
(female), cholesterol (female) and decreased calculated indirect
bilirubin values (females – wide standard deviations) in wk 4 were
cited in the text (p. 51).  An expansion of why values were not
considered biologically significant and comments on wide standard
deviations for some parameters have been added text where appropriate
without historical control discussion.

OK	Page 56

There were toxicologically and statistically significant elevated kidney
weights in male rats at the mid and high dose levels.  The male rat
increases were consistent with the microscopic renal changes associated
with accumulation of alpha-2µ-microglobulin neuropathy.  This induced
alteration is known to be male rat specific and was an expected finding.
	No comment

	The text states that these elevated weights are consistent with
microscopic changes; this is true but should be clarified that
microscopic changes were not performed on the mid-dose animals (except
Table 14 data for one mid-dose male and female rat).  Since only the
high dose group was evaluated, it is uncertain if the induced male rat
nephropathy occurred at all exposure levels.  This was the only
histopathologic finding noted in treated animals.

	Description of histopathology of kidneys indicated that kidneys from
mid dose male rats were not evaluated.  WG had decided that, since light
hydrocarbon nephropathy in male rats was an expected finding in this
series of gasoline-based studies and was not considered relevant to
humans (EPA, 1991), kidney pathology at all dose levels would only be
performed in the BG and BG + MTBE 13 wk studies because these materials
were also being tested in 2-year chronic bioassays.  Extensive
histopathology of male kidneys would only be performed at the
20,000mg/m3 level for other gasoline + oxygenate blends being studied
under the shorter 211(b) program design.  Experience in other light
hydrocarbon studies indicate that increased kidney wt in male rats
usually correlated with microscopically identified light hydrocarbon
nephropathy.  Therefore, the statement in the text that males in lower
exposure levels “would be expected to have shown similar effects in a
treatment–related pattern” is appropriate.  However, since kidney wt
were only statistically significantly increased at 10,000 and
20,000mg/m3, the text statement was qualified to refer to the mid dose
only.  OK	Page 58

The male rat nephropathy exhibited near resolution following the 4-week
recovery period.  Since male rat nephropathy attributed to
alpha-2µ-macroglobulin accumulation is generally regarded not to be
relevant to human risk assessment, these findings also were not
considered in the NOAEL determination. 	No comment

	Results (page 51) mentioned the increased liver weights in male rats at
the two higher exposure levels; however, these findings appear to be
dismissed since they are not mentioned in the Summary (page 8) which
states that besides kidney weight changes, ‘no other organ weight
changes’ were noted.  The liver changes are similar to the noted renal
weight changes observed in that both showed higher values at the
mid-dose than the high dose.  Induction of p450’s by G-ethanol would
be expected.  It is not clear if hypertrophy or increase SER/RER changes
were part of the histopathological evaluations or that changes were
assessed and not seen since this diagnosis were not included in the
histopathological Tables provided.  Induction of p450 and associated
liver weight changes would be expected to be reversed upon cessation of
treatment.  Given the established association with the induction of
p450, liver weight increases and liver cancer, these findings should be
presented in a more comprehensive and consistent manner. 	Text of the
Results section indicates that both kidney and liver weights were
increased in male rats in 10,000 and 20,000mg/m3 groups at terminal
sacrifice.  In both organs, the mid dose increase was higher than the
high dose increase.  When organ wt relative to body wt was calculated
for kidney, the high dose value exceeded that mid dose because terminal
body wt of high dose rats was lower than mid dose; in liver, the
relative wt of mid dose animals remained higher.  There were no
microscopic correlates to these liver wt changes.  Tissues were examined
for hypertrophy, but not for SER or RER changes.  Pathology findings
were reported by exception so it is unlikely that significant liver
hypertrophy was observed.  The study pathologist has been consulted for
verification and agreed with this assessment.   OK

The occurrence of liver wt changes in male rats has been included in
summary (p. 7).  The liver may often increase in weight as a
non-compound-specific adaptive metabolic response to exposure to a
variety of chemicals without representing an adverse effect and is not
used in setting a NOAEL.  Since this is a laboratory study report, the
role of P450 induction in the liver is not addressed.  OK	Page 51;

Page 7

Table of contents page numbers are missing. 	Page numbers will be added
in the finalized report.

	Page 13-14: animals were received prior to protocol sign off by Study
Director?  This is not a standard practice and possibly a SOP deviation?
	Not an SOP deviation although HLS routinely attemps to have the
protocol signed before animals are received.  Not a GLP deviation for
the EPA 211b GLP’s 79.60.  OK

	Does the statement of compliance (page 2) need to note the deviation on
page 45 and state that it had no effect on data integrity/ study design?
	This is adequately documented with impact statement (none) in the
Protocol Deviation section 2.23.  OK	p. 48

The conclusion section (page 8 and 54) only notes the body weight change
in female rats; male rat differences should be noted as well.  Liver as
well as kidney weight changes should be noted.  When setting NOAEL, the
report should make it clear that the male rat nephropathy (and liver
weight changes) is not being taken into consideration. 	Summary and
conclusion sections have been revised to include this information.  OK

	Pages 8 and 59

GFAP Assay:

These studies appear to be properly conducted. Editorial comments from
previous reviews regarding the format, i.e. lack of study specifics,
remain. 	At present, the WG has decided not to require extensive
revisions in presentation from the study investigator, Dr.
O’Callaghan, but will request clarification of statistical methods,
cited by both reviewers as incomplete.  It is also worth noting that Dr.
O'Callaghan is internationally recognized for his work on the GFAP
assay, and the WG has confidence in the integrity of the study
methodology (including statistics) and its conclusions.    	Appendix AA

Pituitary GFAP levels could only be detected in one male per dose group
and not detected at all (0/20) in female rats; due to the paucity of
data and inability to make any conclusions from this region of the
brain, this data should be omitted from Tables and noted in text that
analysis of pituitary could not be adequately performed. 	WG agrees with
the reviewer’s comments and will discuss with Dr. O’Callaghan,
deletion of sparse pituitary data from Tables 5 and 6, but retention of
individual values in Table 7, primarily because this series of studies
is one of the first in which GFAP levels in pituitary have been
measured.  Although other 211(b) reports in the series show somewhat
better recovery of pituitary data, this study has no usable data for
females in any dose group and only 1 entry/dose group for males.  To
include this limited data in summary tables (5 & 6) gives an erroneous
impression that sufficient animals/group responded.  Keeping individual
entries (Table 7) provides an idea of the level of GFAP likely to be
present in this small organ, providing a starting point for improvement
of assay methods. Pituitary data has been excluded from GFAP report
summary tables and text.  OK

	Appendix AA;

Tables 5 ,6, Results pp. 17-18

Note that text, page 18, states that ‘half of pituitaries’ gave
results below level of sensitivity.  This is incorrect according to
Table 7, where all female (0/20) female values were shown to be below
limits of detection. 	Text has been corrected on p 18 to indicate that
no female pituitary GFAP data was obtained above level of detection, and
only 4/20 male rats (1/group) had detectable levels of GFAP in the
pituitary.  Discussion of pituitary deleted from results and
conclusions.  OK

	AppendixAA

pages 17-18

Editorial note: page 17, Results and Conclusion section: test refers to
Figure 1 when it should be Figure 2. 	Figure 2 has been corrected.  OK
AppendixAA

Page 17

I agree with the discussion of study results and conclusions reached in
the Results and Conclusion Section of the report, page 17-18.  This
section accurately notes the slight but statistically significant
concentration related, some areas, i.e., cerebellum and thalamus, did
show increases in multiple dose groups.  Additionally, even though
significant increases were not seen in female rats, all test article
exposed animals displayed increases in all areas as compared to data in
control animals.  A trend analysis was not performed.  Based on all of
the data findings, the report states that ‘a small degree of
damage-induced gliosis does occur in several brain regions’.  I agree
with this statement, even though the response was relatively slight.
There does not appear to be criteria for this assay prior to the conduct
that would label a test article as ‘suspect’ or ‘positive’. 	No
comment

	The summary, page 7, is incorrect and consistent with the conclusion
when it states that “Gasoline Ethanol Vapor Condensate under the
regiments employed does not result in gliosis in any of the nine brain
regions examined”.  This must be changed to reflect the stated
conclusions (as noted above). 	Dr. Goldsworthy is correct.  The
statement in the Summary was a text error and did not reflect the actual
results of this study.  The section has been rewritten to accurately
present the findings of this study in the second draft.  OK	Appendix AA

Page 7

211 (b) Research Group Reviewer

This memo summarizes notes from my scientific review of the first draft
of the BG/EtOH subchronic and satellite studies.  For brevity, issues
that have already been addressed by QA and Drs. Schlesinger and
Goldsworthy, and/or EPA are not included here unless additional emphasis
seems worthwhile. 	Noted.

	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. 	Section 2.4 (test substance) references to Appendix S
where the GC assay data is presented.  OK	Appendix S

13 week Subchronic study:

The EPA reviewer noted that since the GFAP assay showed positive results
in male rat brain at concentrations of 10000 and 20000mg/m3, a NOAEL of
2000 mg/m3 should be considered for the subchronic study, rather than
the 10000 mg/m3 identified.  The GFAP animals were fully part of the
13-week study for the entire exposure period rather than a satellite
groups terminated at a different time.  [A similar issue arose for the
positive results in the GFAP assay of BG/ETBE.]  Assignment of overall
LOAELs and NOAELs will be determined by the 211(b) workgroup. 	It has
been determined by the RG that a summary table listing results of all
studies with NOAEL and LOAEL for each study be included in the
Subchronic toxicity report.  Overall NOEL and LOEL values for the entire
study set will not be determined.

The summary table on p. 9 in draft 2 requires correction for the
Immunotoxicity results.  Positive results [decreased effect in IgM
antibody forming cell (AFC) assay were seen at the highest dose level
making the NOAEL = 10000mg/m3.  NOAEL entry for the subchronic study
should be 10000mg/m3 as presented in the text, rather than <20000mg/m3

Summary table revised	Page 9

Summary

P. 6, 3rd paragraph: Possible rewrite of section referring to large
differences between nominal and analytical concentrations, refer to
comments for Section 3.1 Chamber Monitoring [p. 2 of this memo]

Section has been rewritten.  OK	Page 6

P. 7, 1st paragraph, last sentence:  QA noted a small variation in
week-to week analyses of major components of chamber atmospheres during
exposure 50.  Suggest the last sentence be slightly revised to indicate.
 “Overall, the data were consistent from week-to-week…”
“Overall” leaves space for slight variations in component
distribution in 1 of at least 65 total exposures. 	“Overall” added
to Summary and R&D.  OK	Pages 6 and 52

p.7, 3rd paragraph, line 8:  In accordance with peer reviewer comments,
change entry for reduced body weight gain in females of the 20000mg/m3
group, from “during the latter 12 weeks” to “during weeks 2-13”.

The statement that differences in body weight between air controls and
20,000 mg/m3 rats abated during recovery is somewhat misleading.
Although weight changes were no longer statistically significantly
lower, absolute weight and weight changes remained lower than controls.
Suggest the sentence be revised as:  “The differences in body weight
between Air control animals and the 20000mg/m3 exposed animals abated
but remained lower during the four week recovery.”  [also cited by
Peer reviewers and QA.  See Section 3.5  Body  weight]

Increased food consumption by both sexes during Recovery period should
be reported in the Summary.  [Also cited by Peer reviewers] 	Body
weights and feed consumption sections redrafted to reflect the comments.
 OK	Pages 6-7

P. 8: Include a sentence describing increased liver weights (absolute
and/or relative to body or brain wt.) in males at 10000 and 20000 mg/m3
but that no histopathological correlates were observed [requested by
Peer reviewers].	Done.  OK	Page 7

Methods

p.19, 2.5.6 Weight at Initiation of Exposure:  Please verify that the
mean and range of weight for females in the GFAP study and for males in
the Genotoxicity study match data supplied to subcontractors, as
requested by QA [item 8, Audit dated 3/21/02].

Weight ranges revised as appropriate.  OK	Page 20

p. 23, 2.10.4 Environmental Conditions:  Please verify actual
temperature and humidity with raw data as requested by QA [item 9, Audit
dated 3/21/02). 	Temperature and humidity revised as appropriate.

OK	Page 23

p. 36, 2.16  Immunotoxicity

line 7: please spell sensitized with a z, rather than s. 	Done. OK	Page
37

p. 42-43, 2.20 Statistical Analysis:  Descriptions of additional
neurobehavioral analyses, recommended by EPA, will be added to this
section. 	Done. OK	Pages 45-46

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. 46].  Peer reviewers have requested
confirmation of the number of exposures. 	Not needed here but reiterated
in the Summary and M&M.  OK	Pages 5 and 16 and 25

p. 46, 3.1 Chamber Monitoring:  Considering the wide differences in
nominal and analytical concentrations (16-43%) seen here but not in
other BG/oxygenate studies, more detail explaining methods for resolving
the differences should be provided, as was supplied to EPA in Response
to Peer reviewers [4/23/03].   Revision could be similar to this --
“Ethanol appeared to have significant impact on chamber monitoring
methods.  Use of a Miran calibrated at a wavelength more sensitive to
ethanol may have given a falsely low chamber concentration compared to a
Miran calibrated at a wavelength for total hydrocarbons.  Thus, actual
concentration in each chamber likely fell between analytical and nominal
concentrations and was equal to or greater than target
concentrations.” 	Done.  OK	Pages 49-50

p. 49, 3.5 Body weight:  (Tables 4, 5):  Both Peer reviewers requested
that this section be re-evaluated.  They did not agree that differences
between control and treated rats abated during the four weeks of
Recovery since body wt decreases (both sexes) appeared quite similar at
terminal and recovery sacrifice periods [also cited by QA].  An
alternative paragraph, submitted to EPA [4/23/03] is cited above under
Schlesinger  Section 3.4, p.8. 	Done. OK	Page 53

In the final sentence of text in the draft report, referring to the
absence of weight effects in the Neuropathology satellite rats, please
add that only 5 rats/sex/group were involved. 	Done.  OK	Page 53

p.49, 3.6 Feed Consumption (Table 6):  Add specific entry that feed
consumption was higher for 20000 mg/m3 animals of both sexes during the
Recovery period. [also cited by Peer reviewers] 	Done. OK	page 53

p.49, 3.7 Neurobehavioral Studies:  Any changes in interpretation due to
application of revised statistics requested by EPA will be addressed
(e.g. landing foot splay). 	Done – no significant effects. OK	Pages
54-55

p.50, 3.8 Clinical Pathology:  Study author cites similarity of observed
data to historical control ranges.  Peer reviewers have requested that
where historical control ranges are mentioned, that data be provided.
Historical control data has been reviewed to make comments for the
report but will not be added to report since the concurrent control data
is the most important data for interpretation.  OK

	3.8.1 Hematology and Coagulation (Tables 9 & 10):  Should statistically
significant (P≤0.05) decreases in HGB and HCT in 2000mg/m3 males at
week 13 only be entered here? 	Statistics reviewed and no significance
was indicated at the 2000 mg/m3 level, only at the 20000 mg/m3 level.
OK

	211(b) Research Group QA/QC Reviewer

The draft report (dated December 10, 2001) for this study was reviewed
(2/13-3/19/02) to assure compliance with GLP Regulations (Part 79.60)
and that the report represents the data collected.  One hundred percent
of the text and summary tables of the report were verified and at least
10 percent of individual data were verified.

	No comment needed

	Compliance Statement:  Since it is the sponsor’s responsibility to
maintain the method of synthesis, fabrication, or derivation of the test
fuel, and this has not been completed, it must be included in the
sponsor’s compliance statement.

	Done.  Please make this GLP compliance statement match the Baseline
Subchronic one to include all of the GLP issues found in the Satellite
Immunotoxicity, Micronucleus, and SCE reports.  The Compliance statement
should also indicate that the method of synthesis documentation was not
available during the study, but is now available with the Sponsor.

Compliance statement up-dated.	Page 2

The status of the GFAP laboratory concerning GLP compliance should also
be included in the GLP compliance statement signed by the sponsor.  A
list on non-GLP compliant issues is being prepared for the GFAP report.
These items must be included in the statement.

	Noted but list of non-compliant issues has not been received from QA.
It was decided to leave the terminology ‘spirit of GLP’ and not
indicate non-compliant issues.  The QA Statement for the GFAP report
still needs to be completed.

QA statement completed

	 Page 4, Quality Assurance Statement:  Since HLS has inspected all of
the subcontractors (except CDC) these facility inspections should be
listed on the QA statement for this study (Immumotox and BioReliance
periodic inspections).

	No – as previously stated, our visits were general qualification
visits and not study specific visits and will not be added to our QA
Statements.  It is my opinion that since the GLPs require the Study
Director and the Sponsor both declare that the study was conducted in
compliance with the GLP regulations, every piece of evidence that brings
them to that conclusion should be included in the QA Statement.

QA statement completed to include Subcontractor general inspection
visits.

	 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.

	Done.  OK	Page 5

Page 7, last sentence, first paragraph:  There was a small variation
noted during Exposure 50.  A small increase in Trans-2-pentene and a
decrease in N-pentane content of the atmosphere was noted.

	Noted but this does not need discussion in the Summary.  OK

	 Page 7, third paragraph, first sentence:  Animals were unremarkable in
the chambers…..

	Corrected.  ok	Page 6

Page 19, Weight at Initiation of Exposure:  Please verify the mean and
range of the females in the GFAP study.  They appear in the back of the
GFAP report as a mean of 213.5g and a range of 201 – 230 g.  As well,
the Genotoxicity males ranged from 204 – 254 g.

	Weight ranges have been corrected.  (Note:  Mean body weight for GFAP
females = 213.6g)  OK	Page 20

 Page 23, Environmental Conditions:  Temperature, actual was 21 to 26
degrees C. and relative humidity was 34 to 58%, I believe.  Please
verify.

	Corrected.  OK	Page 23

 Page 29, Calculation (of feed consumption):  The calculation should
indicate the denominator is based on the group mean body weight, if this
is still the case.

	No – the feed consumption is calculated for each animal and then
summarized.  OK

	 Page 31, Temperature, Relative Humidity, Noise Level, Illumination:
The “actual” values listed are those for 508-C.  Should the values
for 508-A be listed as well?  They differ somewhat from the reported
values  (Temp – 22-24 degrees C;  Rel. Hum – 52-86%;  Noise –
62-65 dB; Illumination – 61-66 footcandles).

	Report correct as is; page 31 are ranges for FOB evaluations, while the
values listed here are on page 32 and are for motor activity
evaluations.  OK

	Page 46, last paragraph:  Some indication should probably be given to
detail what measures where taken to explain the differences in actual
and nominal concentrations (flows recalibrated, etc.)

	Done.  OK, don’t remember this being the reason for such differences,
though.

See pp. 51-52; Appendix S   	Pages 49-50

Page 48, first paragraph:  data were consistent from week-to-week except
for exposure 50, where there was a slight increase in Trans-2-pentene
and a decrease in N-pentane content.

	Noted but this does not need discussion in the R&D.  OK

	Page 49, Body Weights:  The next to last sentence indicates that the
differences in weight between the air control animals and the
20,000mg/m3 exposed animals abated during the 4-week recovery period.
This does not appear to be true, as the Group 4 mean body weights were
17g lower than the air control at 12 weeks and 16g lower at 17 weeks.
Please clarify.

 	Done.  OK

	Page 53

Text, Section 3.8.1, Hematology Results, p.50:  In addition to
statistically significantly decreased erythrocyte counts, hemoglobin and
hematocrit values were also significantly decreased in the group 4 males
at the terminal interval.  Should this be discussed in the report text?

	Done.  OK, but doesn’t indicate group 4 males.  Merely indicates male
animals.

Text Revised to indicate Group4.

	Page 55

Page 50, FOB:  The control value for the landing foot splay in Trial 1,
Week 13 appeared unusually small when compared to other female values.
This may also explain why the treated groups were significant.

	Based on new statistical evaluation with averaged values for both
trials, a significant difference is not noted and the Results
&Discussion revised.  OK	Pages 54-55

Appendix A:  Pretest particle size results need to be reported.

	This data has been added to Appendix Q – Testing Room and Chambers
Environmental Monitoring.  OK

	Appendix Q

Page 1

Appendix D, Individual Body Weights:  For Group 4 male 4020 at week 7, a
body weight of 417 grams is reported in the appendix.  The study data
reflect that this animal was reweighed and the body weight determined
and verified to be 455 grams.  The Computer Raw Data Change and Addition
Form shows that the edit to correct the body weight value was made on
June 5, 2001.  However, review of the proof of edit printouts indicate
that the food consumption value was inadvertently edited rather than the
body weight.  The body weight and food consumption data will need to be
corrected to reflect the appropriate values.

	Raw data corrected and report pages reprinted.  OK	Appendix D page 4

Summary tables and statistical analyses will also be affected and should
be corrected accordingly.

	Tables reprinted with statistics as needed.OK	Tables 4-5

Appendix H, Individual FOB Evaluations:  The keys for FOB evaluations
included in the study report and raw data differ slightly in the
presentation of the categories for Abnormal Motor Movements.  The key
contained in the study records includes Abnormal Movements (scores of
none, slight and extreme) under Open Field Evaluations, whereas the
report key presents Abnormal Movements with subcategories of
convulsions, tremors and fasciculation.  In addition, under Reflex
Assessment, Visual Approach, the report key presents separate categories
of 4 (jumps or turns abruptly to avoid) and 5 (attacks and/or bites)
whereas the key in the study records has both of these descriptions
combined as category 4.  All data and report documents should
consistently list these parameters.

	Appendix H preface page revised for consistency with key in data.  The
report key for Visual Approach has been changed but the report key for
Abnormal Movements in the report still indicates the three parameters -
convulsions, tremors and fasciculation.  If the data were not recorded
this way, they cannot be reported this way.

Data was recorded for fasciculation, tremors and convulsions separately;
report is consistent with data.  A note has been added to the key in the
data to clarify what abnormal movements includes and that an entry was
made for each type.	Appendix H pages 1-5

Appendix I, Individual Hematology Values:  Manual differential counts
were performed and reported for the following animals:  Group 2F 2502
(Wk 4), Group 2M 2002 (Terminal), Group 1F 1513 (Terminal), Group 2F
2506 and 2509(Terminal) and Group 4F 4512 (Terminal).  The appendix
reporting differential values should be footnoted to indicate that
manual readings are reported.  Should the manual readings values be
included in the statistics and compared with the automated readings
values?  The numbers of cells observed are greater in the automated
readings; therefore, it may not be appropriate to include the manual
readings of 100 cells in the statistical calculations. Appendix I,
Individual Hematology Values:  The % Reticulocytes were determined and
statistics were performed on these values but these values and
statistics were not reported in the report.  The %B, %E, %L, %M, %N and
%U were also determined and statistics appear in the data with no
inclusion or discussion in the report.

	Note added to preface page for animals with manual differential counts.
 Per SOP, the manual derived values are OK to be included in the
calculations with the automated readings.

Per SOP, only absolute values are reported and not percent values.
Statistical analysis of hematology data automatically prints these
tests.  Corresponding STAT pages have been removed from the data.  OK
Appendix I page 1

Appendix S:  The proportion of Ethanol to other co-elutes needs to be
clearly indicated as it is important that Ethanol be there in a certain
volume. Presently one can only gather this information in the footnote
concerning the extraction efficiency.

	See Section 3 in analytical report: The value of Ethanol is: Total
value of “Ethanol & 3-Methyl 1 Butene” –0.44.  Nothing changed…
still one has to calculate the ethanol value.

Text Revised to indicate 9.03% ETOH value.

	Appendix S, Analytical Report, Page 6:  The second paragraph is
confusing.  It might be clearer to first indicate the problem,
“Subsequent to...”.  Then take a stepwise approach detailing how the
final “All Samples” column was calculated.  First, the coelute was
separated from the ETOH/3-methyl 1-butene peak.  Second the value was
corrected for extraction efficiency.  Third, the other components were
reapportioned accordingly.  It should also be made clear that only the
“All Samples” column is corrected for extraction efficiency and
separation of the coelutes.

	Corrected.  It may be a little clearer than before.  	Appendix S page 6

The analytical method and analyses from EMBSI indicate that 2-Methyl
2-butene will be a component that is found.  Are we sure that 3-Methyl
1-butene is what is coeluting with Ethanol?

	R&D work done prior to study start indicated that Ethanol & 3-methyl,
1-butene coelute.  OK

	Appendix S, Summary Table of Exposures 2-67:  It appears that the
headings of the columns labeled TM-2 and Spiked Control-2 are reversed.

	Correct as is. This was the injection scheme.  OK

	Appendix S, Table IV:  In the data, The sample ID for the ethanol
standard in CS2 appears as SC2.  This is the sample ID of the spiked
control samples.  Please verify that data have been reported correctly
and correct data if necessary.

	Correct as is.  OK

	Analytical data:  Data indicates that both column 5 and 6 were used and
that differences were seen between the two columns (retention times of
16 and 18 minutes for EtOH).  Please verify that the correct column was
designated as they appear incorrect on some days.

	Correct as is. The column info is correct.  OK

	Analytical data:  Certificates of Analyses were not found for all
standards used in the analytical work  (isobutane/n-butane,
isopentane/n-pentane, isooctane, and the new benzene standard not
found).

	These standards are common reagents used in the laboratory and do not
have COAs.   OK

	Also work related to determining the retention time of Benzene needs to
be included with this data.

	Benzene data added.  OK

	Sister Chromatid Exchange Assay:  Table 6:  Errors were found in the
“Range of SCEs Per Animal” Column.  They are:  Animal #1032, Min. 4;
 Animal #1035, Max. 8;  Animal #2024, Min. 4;  Animal #5033, Max, 34.
Please verify these values.

	Table corrected.  OK	Appendix Y page 19

Genotoxicity Report, Food Consumption Appendix:  The Computer Raw Data
Change and Addition Form (CRDCAF) indicates that the food remaining
weight for animal 1032 at week 4 was to be edited from 447.5 grams to
427.6 grams (the original weight was that of the wrong feeder).  The
proof of edit printout indicates that the food remaining weight was
incorrectly edited to 395.1 grams, not 427.6 grams as indicated on the
CRDCAF.  The data will need to be re-edited to reflect the correct food
consumption value.  Summary tables and statistics may also be affected
and should be corrected accordingly.

	Data corrected; all appropriate report pages reprinted as needed.  OK
Appendix X Tables E & I

Immunotoxicity Report:  Tables 1 and 2:  The “less than or equal to”
signs are missing from the table.

	Corrected.  OK	Appendix Z Tables 1-2

GFAP Report, Page 17, section D, Typical Results:  These data need to be
further identified.  Are these comparable strains, ages, sex, etc.?
These data actually have to be trackable to a study.  Are there data
available/compiled to verify this Figure?

	A citation for these results is now mentioned in the Figure Legend and
reference section (Martin and O'Callaghan, 1995).  OK	AppendixAA page
21

GFAP Report, Page 17, Results and Conclusions:  Toward the bottom of the
page it is indicated that ethanol is not as neurotoxic as white spirit,
while previously it indicates that a small degree of damage-induced
gliosis occurred in several brain regions of the male subjects.  Please
clarify.

	Clarified and reference to ‘white spirit’ removed. ok	AppendixAA
pages 17-18

GFAP Report, Page 18, Top of the Page:  It is indicated that half of the
pituitaries in females gave results that were below the level of
sensitivity, when all female pituitaries gave results that were below
the level of sensitivity.  Only one male in each group had a result that
was above the level of sensitivity for this assay.

	Pituitary discussion deleted.  OK	AppendixAA pages 17-18

GFAP Report, Page 28, Table 5:  The thalamus value for Group 1 should be
1.47±0.11 and the SEM for Group 4, Striatum value should be 0.13.
Please verify.

	Values checked and OK as is.  OK

	GFAP statistical report:  Please verify the numbers entered for the
statistical report for the hypothalamus for Group 2, they may not be
correct.

	Values checked and OK as is.  OK

	GFAP Report, Page 28, Table 6:  Group 3, Thalamus value should be
1.77±0.12.  Please verify.

	Corrected.  OK	AppendixAA page 28

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.

	API auditor to provide a listing.  COVERED EARLIER

	GFAP Report, Table 5, Pituitary:  The SEM values should not be given as
there was only one value given in the group.

	Pituitary deleted from Tables 5 and 6.  OK	AppendixAA page 28

DATA

Receipt records for the tanks of test material indicate that two tanks
#10’s were received.  Is that the case?  Were both used?  Please
clarify.

	Second container labeled # 10 was actually # 13; Pharmacy records
corrected.  OK

	The body weight data printout for Group 4 male 4005 at Week 2 shows an
original body weight of 301.4 grams.  A reweigh of the animal resulted
in a weight of 312.7 grams, which was confirmed by a second reweigh of
313.4 grams.  Based on the reweigh data, the animal’s body weight was
edited to 313 grams.  The documentation of reweighing, which is
hand-recorded on the body weight printout, shows the second reweigh
value crossed out with a notation indicating that it was not necessary.
Per SOP, the second reweigh was necessary to confirm the first reweigh
and to justify the body weight edit.  The notations in the data should
be corrected to indicate that the second reweigh was necessary. For
confirmation of the first reweigh.

	Correct as is according to SOP. The original BW was 301.4g, the first
reweigh was 312.7g which is a difference of 11.3 g. According to SOP,
the reweigh (312.7g considered the second value) is correct because it
is between 11-20g. The BW 313.4g that was crossed-out (as it should have
been) is only needed if the first reweigh 312.7g is more than 20g
different from the original BW of 301.4g  (This third weight is
indicated as BW2) Basically, the second reweigh was used and the third
reweigh should not have been taken.  OK

	The same situation as described above in item no. 2 was also noted for
Genotoxicity animal 5031 at week 2.  The data notations should be
modified to indicate that the second reweigh was necessary to confirm
the first reweigh value.

	See response to question 2. There is only 11g difference between the
original BW and the first reweigh. Also, it is in week 1 not week 2.  OK

	Comment:  The Motor Activity data for each set consist of
computer-generated printouts on fan-folded paper.  Specific information
needed to reconstruct study activities (i.e., study no., set no., and
interval) are indicated only on the first page of the printout for each
set.  The perforated pages are beginning to separate and although the
data are currently bound in notebooks, they do need to be removed from
the notebooks in order to view the printed data values.  It is
recommended that individual data page be identified and/or consecutively
paginated to allow for reconstruction of the data package should the
individual pages become separated.

	OK as is.  Electronic record if needed can be reprinted.  OK

Comment:  The Bleeding Lists for Clinical Pathology include a column for
the recording of sample collection times, however the times are not
documented on this form.  Inclusive bleeding times (start to finish) are
documented on the form entitled “Animals for Laboratory Studies and
Sample Transfer Form”.  It is suggested that the Bleeding List be
annotated to indicate where bleeding times can be found.

No in life individual bleeding time recorded, only start and finish. See
clin path SOP.  OK

	Statistical Analyses:  DBILI values were apparently analyzed by
Fisher’s Exact analysis when 75% of the values within the data set had
the same value.  This test is not included in the statistical methods
presented in the study protocol.  Should the protocol be amended to
include this analysis?

	Fisher’s Exact analysis added to report.

The protocol was amended.  OK

	Individual Gross and Micro Table:  Frequently there are indications of
recuts (larynx particularly) with notations that the recut section was
no improvement over the original.  These tissues are counted as having
been reviewed.  Is this SOP?  As long as there is some unremarkable
tissue present, even though it may not contain all of the required
elements, the tissue is reported as normal and accounted for?  Please
comment.

	Evaluations performed per SOP.  OK

	In the GFAP data book, it is indicated that Christine Mason is the API
Principal Investigator.  This should be corrected.   Also Amendment #2
is present in the data book, but # 1 is not there.  It should be kept
with the data.

	Subcontractor data correctedOK

	The GFAP assay for the Rest of Brain samples:  It is not clear why the
1:400 dilution was not reported.  Some brief explanation should be given
for not using this dilution.

	Multiple sample dilutions read off the same curve are typically used
with the dilution putting the values in the middle of the curve being
optimal.  For this particular brain area for this experiment the 1:400
was not used because the 1:200 values fell on the curve.  OK

	GFAP data:  For animal numbers 38 and 39, please check the data sheets
indicating dilutions and calculations used.  It appears that the correct
values were reported.  Also please check #20, cerebellum values.  It is
indicated to use the 1:200 dilution, but that value and another dilution
value are very similar.

	Data sheets have correct numbers.  OK

	GFAP Report:  There appears to be a difference in rounding or recording
in the BCA assay between the plate reader, the print-out and the
summarization sheet.  A comment explaining this should be included in
the data as presently it is unclear how this could happen.

	Calculations (values per standard curve & rounding) done by validated
computer program (under GLP).   OK

Micronucleus Satellite Report

Comments	Action/Response	Page #

EPA 	See page 2 of response to comments

	Peer Reviewer - Schlesinger

MICRONUCLEUS SATELLITE STUDY.

Were there any gender differences noted?  If an appropriate statistical
test was used, then results from both genders could have been
statistically evaluated at the same time, which would not have reduced
the power of the analysis as implied.

	Data was evaluated for each sex separately and combined and no overall
effect was seen.  Individual data supplied on Table 2  OK	Appendix X,
Table 1, p. 19 9avg for each sex); Table 2 pp20-21

Table 1 – There should be some error terms (SE or SD) provided with
the group means for the % and incidence datasets.

	SD values added.  OK	Appendix X pages 18-19

Peer Reviewer – Goldsworthy

Study was properly conducted and scientifically sound; not study
deviations were noted.  Positive and negative controls performed as
expected and within historical range.

	Noted.

	Results section (page 15) should refer to Tables 1 and 2.

	Done.  OK	Appendix X page 15

I agree with the conclusions that Gasoline Ethanol Vapor Condensate did
not induce micronuclei under these experimental conditions.

	Noted.

211 (b) Research Group Reviewer

Please add a table of male and female data separately to be consistent
with the second draft of the Gasoline study.  No additional statistics
are necessary.

P. 15, Results:  Please enter Tables 1 and 2 next to the Micronucleus
Test heading  [requested by Dr. Goldsworthy].

	Done. OK

Added to the 1st paragraph of this section. OK	Appendix X page 19

Appendix X page 15

P. 18, Table 1:  Consider providing error terms [SE pr SD] with group
means for percentage and incidence data sets.  [requested by Dr.
Schlesinger].

	Done. OK	Appendix X pages 18-19

211(b) Research Group QA/QC Reviewer	See page 29 of response to comments

Sister Chromatid Exchange Satellite Report

Comments	Action/Response	Page #

EPA 	See page 2 of response to comments

	Peer Reviewer - Schlesinger

SISTER CHROMATID EXCHANGE SATELLITE REPORT.

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.  Furthermore,
the paragraph describing the criteria for 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”.

	A trend test was added to the statistical methods.

[in accordance with EPA recommendations]

OK	Appendix Y page 11

The actual results are presented at the end of the last paragraph of the
Results and Discussion section.  There is no really no discussion
presented.  Furthermore, the 1st paragraph of Results and Discussion
should be in the Introduction while all of the other paragraphs until
the last half of the last paragraph on the next page should be in the
Methods section.

	Report OK as is – all information is available to the reader although
not in the order that the reviewer would prefer.  OK

	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 Ethanol
Vapor Condensate was negative for the induction of SCEs in rat
peripheral lymphocytes.

	Noted.

	Comments include:

(1) Study title should include “Gasoline Ethanol Vapor Condensate”.

	Test substance indicated on page 1.  No change  OK	Appendix Y page 1, 6

(2)  Suggest noting in the Summary (page 8) that the positive controls
fulfilled the requirements for a valid test.

	Not necessary but noted in Conclusion on page 12.  OK	Appendix Y page
12

211 (b) Research Group Reviewer

p.11, Evaluation of test results, 1st paragraph.  Revise sentence in
Statistics to include the one-tailed Dunnett’s test and trend test
requested by EPA.

	Done. OK	Appendix Y page 11

p.12, Results, 4th paragraph:  Revise sentence describing Dunnett’s
test to reflect one-tail analysis and trend test.  EPA revised
statistics did not alter negative results for BG/EtOH.  The reference to
Dunnett’s test in the Summary should also be adjusted.

	Done.  OK	Appendix Y page 12

p.19, Table 6:  QA indicated a discrepancy in Range of SCEs per animal
for Animals 1032, 1035, 2024, 5033.  Please check against raw data.

	Done. OK	Appendix Y Table 6; page 19

211(b) Research Group QA/QC Reviewer	See page 29 of response to comments

Immunotoxicity Satellite Report

Comments	Action/Response	Page #

EPA 	See page 2-4 of response to comments

.

	Peer Reviewer - Schlesinger

Immunological Evaluation of Gasoline Ethanol Vapor Condensate in Female
Sprague Dawley Rats.

A repeat of methods is not needed in the footnotes to Tables 1 and 2,
Table ES1 is not necessary and does not add to the report.  On page 16,
it is noted that when evaluated as specific activity, the response was
suppressed 85% and 86% when evaluated as total spleen activity.  This is
not clear, and the fact that these are maximum values should be noted.

	The table format is consistent with previous studies conducted.  We
have noted on page 16 that the values are maximum values.  Done.  OK
Appendix Z page 16

 It is noted, rightly so, that there is a significant dose related
decrease in IgM antibody forming cell response to T dependent antigen
and that the decrease reached statistical significance at the highest
exposure level.  Thus, in this case, because the greatest effect was at
the highest level, much more weight seems to be placed on the effect
than for other endpoints noted above in other aspects of the study, in
which effects were seen at the lowest, but not the highest, level.  This
relates to comments noted above about the toxicological significance of
responses that may not seem to follow a classical exposure
concentration-related pattern.

	We concur.  The only effect was on the AFC; therefore, more emphasis
was placed there. OK	Appendix Z pages 8 and 16

Peer Reviewer – Goldsworthy

The study evaluated the potential of four weeks of exposure to G-Ethanol
to effect the humoral immune component of the immune system when
evaluated by antibody-forming cell response to T-dependent antigen sheet
erythrocytes.  This study was conducted properly and was scientifically
sound.

Gasoline Ethanol vapor condensate did result in a dose-related decrease
in the spleen IgM antibody-forming cell response to sheep erythrocytes.
The positive control CPS resulted in the anticipated results.

	No comment needed

	Data demonstrate that G-Ethanol did not affect terminal body weights,
spleen or thymus weight.

A recommended revision in the test is to include the statistically
significant decrease in body weights in CPS group when discussion body
weights alterations in the Results section on page 13.

	CPS is not used as the positive control for body weight.  On occasion
it will produce decreases reaching the level of statistical
significance, as was observed on this study.  Since it was not used as
the positive control for this parameter (dosing regimens and the
duration of exposure were different) it is of little value to address
the weight change of CPS when it was not intended to be the positive
control for body weight.  Accepted  OK

	Editorial note: Table of Contents refers to Appendix ‘B’ when the
Appendix is titled Appendix 2.  (Also eliminate “page 23” on
Appendix 2 cover page.)

	Corrected. OK	Appendix Z page 7

The conclusion that, under the experimental conditions of this study,
G-Ethanol did adversely affect the functional ability of the humoral
immune component of the immune system accurately reflects the study
results.

	Noted.

	211 (b) Research Group Reviewer

p.12, Methods:  Suggest that the last paragraph on p. 12  that describes
the assay very clearly be moved to the Introduction (p. 10).  It would
fit very nicely as the 2nd paragraph there.

	We disagree.  We do not think it belongs in the introduction.  It
belongs in the methods section.  Dr. White disagreed with this
suggestion and text was not changed.  OK

	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.

	If one sex is to be evaluated, ImmunoTox, Inc. concurs with the use of
females because of a more robust immune response.

ndicates a p≤ 0.05 significance in the trend test for relative spleen
wt.  The discrepancy between Table 1 and text requires clarification.
Also check conclusions (p. 17) where spleen wt is addressed.  [also
cited by EPA—were positive control spleen data included in trend
tests?]

	See earlier response [EPA checklist p. 3]  OK	Appendix Z, p. 15

p.15, EPA inquired whether viabilities of 84% were considered low or
correlated favorably with historical values from Dr. White’s
laboratory be provided to address this inquiry?

	See earlier response. [EPA checklist p. 3]	Appendix Z, p. 15

p.16, Results: Dr Schlesinger requested a revision of the end of 1st
paragraph to emphasize that the 85% and 86% suppression of spleen
activity at 20000mg/m3 were maximum effects.  Please revise the sentence
if appropriate.  I realize that the heading “Maximum effect” is
included in Table ES-1.

	The following bolded words have been added to the sentence : "When
evaluated as specific activity the response of the high dose group was
suppressed 85% and 86% when the high dose group was evaluated as total
spleen activity."  	Appendix Z page 16

p.17, Conclusion:  The EPA reviewer discussed the effect of ethanol in
immunotoxicity fairly extensively in the comments, since effects here
were possibly attributable to EtOH content in vapor condensate.  Request
that Dr. White review the EPA comments and add additional information to
the report if practical.

Tables 1 & 2:  The “ ≤” are missing from statistical significance
entries.

	The < was added.  OK	Appendix Z Tables 1 & 2

211(b) Research Group QA/QC Reviewer	See page 29 of response to comments

GFAP Satellite Report

Comments	Action/Response	Page #

EPA 	See page 5-7 of response to comments

	Peer Reviewer - Schlesinger

GFAP levels in Specific Rat Brain areas following a 13-wk whole body
inhalation exposure to Gasoline Ethanol Vapor Condensate:

No specific comments	Noted.  Dr. Schlesinger considered the GFAP report
to be extremely well written

	Peer Reviewer – Goldsworthy

No comments	Dr. Goldsworthy recommended significant changes to the
report format for all GFAP studies but the 211(b) workgroup decided to
leave the format as written by Dr. O’Callaghan.

OK

	211 (b) Research Group Reviewer

p.7, Summary:  Unfortunately EPA received the draft report with the
incorrect 1st paragraph stating that no effects on GFAP were seen with
exposure to BG/EtOH.  Please be sure that the 2nd draft is correct.  The
EPA reviewer recommended a NOAEL = 2000mg/m3 based on the elevated
levels of GFAP in male brain regions, and referred this NOAEL back to
the 90 day study where the NOAEL was set at 10000mg/m3, since GFAP was
performed on animals exposed for the entire 90 day study duration, not
as a shorter exposure satellite group.  The significance of the GFAP
changes for the BG/EtOH study should be discussed with the 211(b)
Working group, sponsor.  This is the only study with positive GFAP
effects.

	Done.  OK	Appendix AA, p. 7

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.”

	Done. OK	Appendix AA, p. 8

p.17, Statistics:  A better explanation of statistical methods for GFAP
has been consistently requested by peer reviewers and now EPA.  Since
the EPA reviewer performed statistical analyses on the male rat
cerebellum data from this study [see EPA comments, 6/9/03], it seems
particularly appropriate to expand the statistical section in this study
at least.

	Done.  OK	Appendix AA, p.17

Although it has been decided by the Working 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.

	Noted.

	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……”

	The 211(b) WG considers the report format adequate as is.  OK

	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.]

	The 211(b) WG considers the report acceptable as is. OK

	RESULTS and CONCLUSIONS:

In the 5th line, the Figure designation for levels of GFAP across
different brain regions is incorrect.  It should be Fig. 2, not Fig. 1.

	Corrected. OK	Appendix AA, p. 17

Delete sentences concerning effects of White spirits on GFAP and
comparison of EtOH and white spirits on GFAP activity.

	Done. OK

	Appendix AA, p. 17

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.

	Done.  OK	Appendix AA, p. 17

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.   Data were only
available for 1/5 males in each dose group and no females.  This data
should not be included in Table 5 which presents mean GFAP levels – a
mean cannot be calculated with data from only one animal.  The pituitary
data could be included in Table 7 individual data, but omitted from
Tables 5 and 6, Mean GFAP levels in Male and Females, respectively.  As
has been seen in other studies in this series, females seemed to provide
much less data than males.  [also been cited by Peer reviewers and QA].

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W

X

w

B*

欀卤

欀恤

B*

B*

H*

B*

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 the individual tables with explanation. OK

Appendix AA,

Tables 5 &6, pp 28, 29

Results, p. 17

211(b) Research Group QA/QC Reviewer	See page 30 of response to comments

211(b) Toxicology Research Group

Ethanol Subchronic Toxicity Study Report

Reviewer Checklist

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211(b) Toxicology Research Group

Ethanol Subchronic Toxicity Study Report

Reviewer Checklist

211(b) Toxicology Research Group

MTBE Subchronic Toxicity Study Report

Reviewer Checklist

211(b) Toxicology Research Group

Ethanol Subchronic Toxicity Study Report

Reviewer Checklist

211(b) Toxicology Research Group

MTBE Subchronic Toxicity Study Report

Reviewer Checklist

211(b) Toxicology Research Group

Ethanol Subchronic Toxicity Study Report

Reviewer Checklist

211(b) Toxicology Research Group

MTBE Subchronic Toxicity Study Report

Reviewer Checklist