Document ID: EPA-HQ-OAR-2003-0065-0453
Agency: epa
Document Type: Supporting & Related Material
Title: 
Posted Date: 2008-04-11T04:00Z

Comments	Action/Response	Page

EPA 

In general, the NHEERL reviewers concluded that the studies were
conducted in accordance with the testing guidelines, once changes
detailed in the API peer review response are made.	No action needed

OK

	Additional comment is requested on the following points.

A statistically significant difference in liver/body weight ratio was
seen in the 20,000mg/m3 exposure group. This effect occurred in the
high-dose treatment group and thus appears to be treatment-related.	This
increase was only seen in males.  Absolute liver wts of both sexes and
female relative wt were comparable to controls.  Additional text has
been added to Results and Discussion OK	37

As pointed out by the peer reviewers' comments and the study group
response, differences were observed in the prostate weights and weight
ratios of all treatment groups. The study group, however, disregarded
this outcome and concluded that the reproductive NOEL was 20,000mg/m3.
Increases in accessory sex organ weights are used as a marker of
androgen action.  Since statistically significant changes were seen in
all treatment groups as either absolute weights or weight ratios, a
reproductive NOEL for this endpoint cannot be concluded from this study.
Therefore, we propose that the lowest exposure concentration employed
(2,000 mg/m3) be considered as a LOAEL.	Additional text has been added
to Results and Discussion.  Because intergroup differences in prostate
weight are seen without a dose response and they are not consistent with
other endocrine-sensitive organs, even in the same animal and further,
there are no histopathological findings, it is considered inappropriate
to adjust the NOAEL based on these intergroup differences.  The
reproductive NOAEL = 20,000mg/m3 has replaced the NOEL cited in the
first draft. 

Including reference to results of the 13-week study (90 days there vs.
105 days here see response to Dr. Schlesinger below) is helpful since no
significant increases in prostate weight or histopathologic changes were
reported in the companion 13-week study.	38

7 

Peer Reviewer – Schlesinger

The study was conducted in a scientifically sound manner and followed
the appropriate protocols. There were no significant deviations from
these protocols that would have affected the outcome of the study. The
investigators conclude that the NOAEL was 10,000mg/m3. However, as noted
below, this reviewer feels that a significant effect on prostate weight
occurred even at the 2,000 level. Thus, the investigators need to better
justify their conclusion in this regard.	Dr. Schlesinger is correct that
the prostate weight changes should not be discounted Additional text has
been added to Results and Discussion. The discussion of intergroup
differences in prostate weight provided above in the response to EPA for
reproductive NOAEL apply here.  Although these differences may result in
the inability to establish a NOEL, it is considered inappropriate to
adjust the systemic NOAEL from 10000mg/m3 based on these observations.
OK

Increases in prostate absolute weight and weight relative to brain
weight are statistically significant at the low and high doses, and
elevated but not statistically significant at the mid dose.  When
prostate wt are compared to final body wt, statistical significance was
observed at all three exposure levels and demonstrated a plateau effect
across 3 levels; this significance was not unexpected since terminal
body wt of male rats showed a slight (not statistically significant)
exposure-responsive decrease.  No microscopic abnormalities of the
prostate gland were observed which would correlate with increased organ
weight (p. 39, Table 23; Appendix Z).  In addition, no significant
increases in prostate weight or histopathologic changes were reported in
the companion 13-week study. OK	38,

Table 23, Appendix Z

General Comment

The exposure group numbering is not consistent in the report. In some
places this is given as I, 11,111, IV and in others as 1,2,3,4.	This
inconsistency was brought to the attention of the study director and,
where practical based on computer system limitations, adjustments were
made.  However, in computer generated tables and compilation of raw data
the number format is predetermined.  This observation does not affect
the validity of the data. OK	various

p. 29. 2.15.1.

Justification for the use of two levels of significance is needed. Also,
there only seems to be one additional test used, the Dunnett, and not
"tests" as indicated in text. 

Finally, it is noted that statistical testing was not done when the
standard deviation was zero. The investigators should have examined
other tests that could have been used in such cases.	These levels of
significance were pre-selected since they were inherent in the
laboratory’s computer statistical package.  Comparison at two levels
of significance has been common practice in this laboratory.

Only Dunnett’s test was used to compare group means to control means
of continuous data.  The report text has been clarified. 

This is a standard statement for the statistical methods section.  In
this study, Table 1 Summary of Survival and Pregnancy provides examples
of where the n is 0 or 1 and this statement is relevant.  These events
are also observed in macro/micropathology incidence data.  No
statistical analyses are applied. OK	29

p. 33. 3.1.

The explanation given for the difference between the measured and
nominal concentrations involves assumed inaccuracies in the calibration
of chamber airflow and the IR monitor. There is no reason why the IR
monitor in particular should not have been accurately calibrated using
accepted procedure. A similar comment applies to the airflow measures.

What accounts for the particles in the chambers, especially the control
chamber.  Was a filter used to clean the incoming air and if not why
not?	

To be precise, the report stated that the ratio of the measured to
nominal concentration was not 1:1, and speculated that the cause MAY
have been due calibration error. In any event, the inaccuracies between
nominal and actual analytical concentrations were slight, and the
analytical concentrations were no more than 1.5% higher than the target
concentrations..  The RG feels confident that the reported chamber
concentrations accurately reflect the atmospheres to which the animals
were exposed.  Text has been clarified OK

With regard to the presence of particles, the RG notes that the mean
particle concentrations were comparable between the control and
treatment group chambers. The primary purpose of making these
measurements in a study of this nature (high vapor concentrations) is to
make certain that vapor condensation is not occurring and to preclude
the possibility of aerosol formation. The results provided in the report
confirm that gasoline + ETOH exposure was to vapors and not aerosols.
Some particles are invariably present in chamber atmospheres,
representing background levels. In fact, careful analysis of the data in
the table on page 34 indicates that the particle concentrations in all
of the chambers including controls ranged from about 4 to 5
micrograms/cubic meter of air with the exception of the low dose group
(2000mg/m3) where total mass concentration was 8mg/m3.  For comparison,
it has been reported in EPA's PM 10 Criteria document that annual
background PM 10 levels in eastern cities range from 5 to 11 micrograms
/cubic meter of air. Thus, the RG believes that the particles in the
chambers reflect ambient background values. OK	

32

33

p. 38. 3.4.2 and Table 21.

There appears to be an increase in prostate weight that is relatively
consistent at all exposure levels even though it does not reach
statistical significance at the 10,000 level. This is discounted by the
investigators since it did not occur in what is termed a
"treatment-related pattern." However, to this reviewer, this seems to be
a real effect even though it did not follow a standard dose-response
profile. There are instances where an effect will be observed that
reaches a plateau even with increasing exposure concentration. Thus, the
investigator's logic that resulted in discounting this effect is
potentially flawed.	

See response to EPA and Dr. Schlesinger’s first comment.  This issue
was also addressed by Dr. Goldsworthy and the 211(b) scientific
reviewer.

Additional text has been added to Results and Discussion OK	

38

Peer Reviewer – Goldsworthy

The study was scientifically sound and was properly conducted.  Exposure
were properly conducted and adequate to test the potential reproductive
toxicity of Gasoline Ethanol Vapor Condensate.  None of the protocol
deviations noted would be expected to adversely effect study results or
data integrity.	No action needed OK

	No effect of treatment was noted on survival, clinical observations,
body weights, estrus cycles, mating, fertility or gestation indices, or
on other reproductive parameters up to parturiton.  An exposure-related
decrease in body weight gain was observed in high dosed male rats during
the pre-mating interval.  No treatment differences were observed with
pregnancy rates, duration of gestation and percent of females completing
delivery.  No difference between treated and control groups were noted
in all parturition parameters, including total number of pups delivered,
viability and lactation indices, the number of implantation sites per
litter, the pup sex ratio, and the number live pups per litter. 
Additionally, sperm count, motility and morphology was not altered by
treatment.	No action needed OK

	There were no exposure-related differences in macroscopic postmortem
assessments.  Increased kidney weights were observed in male rats
(parental generation) at all exposure levels; this is consistent with
the expected induction of hydrocarbon nephropathy.  Microscopic
evaluation was not performed to confirm this observation.  Hydrocarbon
nephropathy is generally not considered to be relevant for human risk
assessment.  There were statistical differences noted in prostate organ
weights in low and high dose treatment groups; these differences were
considered not attributed to the test article because of the small
absolute differences noted and the lack of a treatment-related pattern. 
There were no exposure-related differences in body weights, macroscopic
postmortem evaluations and organ weights in the lactating pups from Air
Control animals.	No action needed OK

	In summary, a NOAEL of 10,000 mg/m3 was determined based on the
decreased body weight gain in the 20,000 mg/m3 exposed parental male
animals.  There was no effect at any exposure level on reproductive
performances.  This, with respect to reproduction performance, a NOEL of
20,000 mg/m3 was concluded.  I concur that these data support these
conclusions.	No action needed OK

	The statistical differences in prostate weight (as noted on page 38 and
in Tables 21) should be noted and commented on in the summary section
(page 6, bottom or page 7 top) when discussing organ weights.	See
response to EPA and Dr. Schlesinger’s first comment. Additional text
has been added to Results and Discussion It was agreed between the WG
and contractor that because the prostate weight differences should not
alter the NOAEL it was not appropriate to cite them in the Summary. OK 
38

211 (b) Research Group Reviewer 

General Comments

The report should have sequential numbering in it's entirety. There are
multiple pages that are identified as "Appendix A, page 1 " including
Appendix A page 1, Appendix A's Appendix A page 1, and Appendix A's
Table II, page 1. This is unnecessarily confusing. Maybe this has
already been changed for other reports.	Final report will be fully
paginated from cover to cover OK

	Specific Comments

P. 5, Summary, 4th paragraph.

I suggest changing the word, "reasonably" to "acceptably," since there
should be SOP or protocol specific criteria for concordance of nominal
and analytical concentration measurements. I recommend this change later
in this paragraph also on p. 6.	Revised to “acceptably” OK	5, 6

Suggest deleting the sentence beginning: "The exact cause of the
differences ." as unnecessary.	Deleted OK	5

P. 14, 2.4.7, Analysis.

Please indicate that the purity analysis of the test substance is
included in the report.	

Added to section 2.4.3 OK	

14

P. 15, 2.4.9, Dispensing.

The test substance was provided as follows, from a reply that I received
from Mike Henley:

The contract is with Global Technology Company sub-contracting Chevron
Research and Technology Company to provide the test substance.

Please correct the supplier to either Chevron Global Technology Company
or Chevron Research and Technology Company, whichever QA considers to be
most appropriate.	Revised to “Chevron Global Technology Services
Company” as per other reports. OK

	15 

P. 18, 2.9.4, Feed Analysis.

typo: "feed" should not be followed by a comma.	Corrected OK	18

P. 19, 2.9.9, Temperature.

The low end for the actual range is quite different than the desired
range. I think this should be described by date in the protocol
deviations.	 This was a transient difference that is already noted as
not affecting the study. OK	19

P. 30, 2.1 5.2, Sperm Motility Analysis.

typo grammatical error: "Statistical were performed.. ."	Corrected to
‘statistical analysis’ OK	29-30

P. 33, 3.1, 1st paragraph.

At 2000 mg/m3, H-7 was 137% of the reference standard. Is this within
the criteria for even distribution?	This value was reassessed on the
following day and was within 5% of target OK	32, Appendix A

Please delete "at least" from the second sentence. All of the readings
were 20%, not greater (if I am in error here, please provide
correction).	Corrected OK	32

3rd paragraph (below table) .

As previously, I suggest changing the word, "reasonably" to "acceptably"
regarding concordance between nominal and analytical chamber
concentrations. I also suggest deleting the sentence beginning, "The
exact cause of the differences ...." as unnecessary. If the differences
were within an acceptable range, there is nothing to explain.	Corrected
OK	32

Please see comment above regarding Appendix A.	This value was reassessed
on the following day and was within 5% of target OK	Appendix A

P. 34, last paragraph.

I suggest changing the word reasonably to satisfactorily or acceptably.
Changed to “acceptably” in last paragraph, first sentence OK	33

P. 35, 3.2.2, Physical Observation Data.

Appendix E, gestation data, includes data for the nonpregnant females;
it should not.	These non-pregnant females were considered pregnant based
on plug or smear data are therefore included in this table. Appendix E
should be footnoted for this, and/or the individual females that were
not pregnant should be noted as such (either option is OK)	34, Appendix
E

3.2.3, Body Weights - Premating.

Male data should have both the total premating period body weight gain
and the total (days 0-105) body weight gain data value in Table 5.	Table
corrected [now Table 6] OK	Table 6

P. 36, 3.2.5, Feed Consumption.

Please indicate that the statistically significant interval was for
group 4 females, day 28-35.	Revised OK	35

3.2.7, Estrous Cyclicity Data.

The control group includes one female with a single 17 day cycle
(#1561), and another with a typical 4 day cycle and a 13 day cycle
(#1557). Both of these had extended diestrus, which as you point out in
the text is indicative of pseudopregnancy. Another female in this
situation is #2568, with 15 day and 4 day cycles. These values appear to
be artifactual rather than biological, with the potential to skew the
group mean. Please respond as to whether one or more of these animals
should be excluded from the group mean for statistical comparison
between control and exposed groups.	The periods of pseudopregnancy were
excluded from calculated values.  Entry added to the preface for Table
14 OK – Appendix P shows the excluded values clearly (nice job!)

	35, Table 14

3.2.8, Mating, Fertility, and Gestation Indices.

Table 15 has the endpoint, Females with defined day 0 of gestation.
However, this is really the insemination date, as a few of these animals
never delivered. Is it possible to footnote the table, or change the
description, to better reflect the data shown in this row?	Table has
been footnoted. OK	Table 15

P. 37, 3.3.1, Pup Body Weight Data.

There is no appendix for pup body weight gain.	This is not possible due
to TASC computer system limitations.  TASC does the weight gain
calculation but does not generate a table of the individual data. OK :o(

	P. 38, 3.4.2, Parental Organ Weights.

Although the increase in prostate weight was only dose-responsive as a
ratio to body weight, all dose groups were elevated (2K and 20K
statistically significant). I think that the explanation for not
attributing these differences to exposure would benefit by commenting
upon the usual range of weight for this organ at this animal age.	

Additional text has been added to Results and Discussion.  Summary of
control data from other 1-2 generation studies in this program were
reviewed and discussed with WG without being added to the text.  No
other BG+oxygenate study showed similar prostate weight changes.
Including reference to results of the 13-week study (90 days there vs.
105 days) is helpful since no significant increases in prostate weight
or histopathologic changes were reported in the companion 13-week study.

38

Page 39, 3.4.4, Macroscopic Pup Evaluation.

Appendix S and U, as well as W, include pup observations for
stillborn/dead, and these should be referenced here. Stillborn data is
also in Table 16.	Table 16 was previously references in appropriate
sections of Results & Discussion [see Sect 3.2.9].  Appendices
references updated OK	36, 38

It is important that these various sources of data match. For example,
Table 20, p. 2, lists 5 stillborn for Group 2, but only 3 as stillborn
from the lung floatation test. Appendix S lists 5 stillborn, Appendix U
lists 5 stillborn. There is no explanation in the table as to why these
other two pups are not identified by the lung floatation test. The
individual data indicate "postmortem changes obvious at the time of
necropsy." I assume that the SOP considers Day 0 nonviable pups that
cannot be tested to be stillborn, but shouldn't that outcome be listed
as a third choice in Table 20? The same query exists for Group 4.	Added
to sect. 2.13.2:“If the lung flotation test was not conducted, then
the pup was categorized as stillborn.” OK	36

Table 3, page 1.

group 4 data includes observations for nonpregnant animal	The
nonpregnant females were considered pregnant based on plug or smear data
and are therefore included in this table OK if footnoted.	Table 3

Table 5.

Males should also have total (days 0-105 body weight gain data.	

Table 6 corrected  OK	Table 6

Table 6, page 3.

Male data should show: day 84-91, 91 -98, 98-1 05, and 0-1 05	Table 6
corrected OK	Table 6

Table 14.

Please see comment above for p. 36. The control data seems to be unduly
influenced by two cycles that appear to be pseudopregnancy.	

The periods of pseudopregnancy were excluded from calculated values. 
Entry

added to the preface for Table 14 OK	

Table 14

Table 15, page 1.

please see comment for 3.2.8, page 36.	Table has been footnoted OK	Table
15

Table 21, page 4.

should list female body weight on the table for absolute organ weights,
just as done for the males.	Added to Table 21, page 3. OK	Table 21

Appendix A, page 6,2.3.2.

typo: "characterized" (no d at the end)	Corrected couldn’t find on
recheck, so assume OK	Appendix A, p. 7; 2.3.2

Appendix E.

The data for the nonpregnant female appears to be included in
gestational observations (#4565).	The nonpregnant females were
considered pregnant based on plug or smear data and are therefore
included in this table OK as long as it’s footnoted	Appendix E

Appendix N.

Food consumption for GD 0-20, which appears in Table 12, should also be
presented.	Added OK	Appendix N

Appendix O.

Food consumption for LD 0-28 should also be presented.	LD1-28 is
presented since feed consumption per protocol was not measured on LD0 OK
Appendix O

Appendix P.

There are three females with estrus cycles that may be artifactual
rather than biological; please review. These are for animals 1557, 1561
(both controls), and 2568 (2000 mg/m3). If these are likely to be
artifactual, they should not be used to calculate the group mean value.	

The periods of pseudopregnancy were excluded from calculated values.
This information is added to the preface for Table 14 OK	

Table 14

211(b) Research Group QA/QC Reviewer

The following items are comments or require further consideration:

Draft Report, Page 2, Compliance Statement.

Since it was the sponsor’s responsibility to maintain the method of
synthesis, fabrication, or derivation of the test fuel, and this had not
been completed during the study, it must be included in the sponsor’s
compliance statement.	Added OK, but needs to match the other studies.  I
believe the statement indicates that the ‘information was not
available during the study, but is now available with the Sponsor.’	2

Draft Report, Page 4, QA Statement.

Is the first entry of Subcontractor Facility Inspection, a PAI
inspection? Please clarify and add PAI’s facility inspection to the
list, if this is not referencing PAI.	Added - OK	4

Draft Report, Page 14, Section 2.4.7, Analysis.

The last sentence in this paragraph indicates that a purity was done
4/27/01 after the start of study 6127. This was well before the start of
this study, so it is not necessary here.	Deleted - OK	14

Draft Report, Page 16, Weight At Initiation of Exposures.

The female weight range should be 112-175 not 185.	Corrected - OK	16

Draft Report, Page 20, Air Changes.

The raw data documents the air changes in Room 708 as 14.1 and in Room
809 as 14.7. Therefore, the actual range in the report should be 14.1
– 14.7, not 12.6 – 14.6 per hour.	Corrected - OK	20

Draft Report, Page 23, Postmortem, Parental Animals (Po), Necropsy
Information.

The first sentence is confusing. All parental animals were necropsied
(26/sex/group). Even if the animal died before its scheduled 3
sacrifice, a necropsy was performed. The sentence should state all
animals necropsied.	Corrected - OK	23

Draft Report, Page 34.

The total mass concentration for group three should be 4.25 x 10-3.
Corrected - ok	33

Draft Report, Page 35, Body Weights, Premating Period.

The first sentence states “an exposure-related decrease in body weight
gain was seen in the 20000 mg/m3 male animals”. This statement should
be clarified since this level was only statistically significant for the
days 0 to 7 and 14 to 21. The decrease in weight gain was slight or
comparable to the other treatment groups. The body weight values for the
2000 mg/m3 do show an exposure-related decrease, not the body weight
gain values This correction should also be made in the report
Conclusion, Page 40, and Summary, Pages 6 and 7.	Summary and Results and
Discussion clarified that body weight gain was decreased in the
20,000mg/m3 animals during the initial 3 weeks of the study.  OK	34

39, 7

Draft Report, Page 36, Feed Consumption, Premating Period.

The second sentence states that a statistically significant difference
was noted at one interval. Table 11 reports two intervals as
statistically significant – Group III Day 0 and Group IV Day 35. This
section of the text should be corrected to agree with the table.	Day 0
is the end of the pretest interval.  There fore, Results and Discussion
clarified that “A statistically significant difference was only noted
at one interval (days 28-35 for the 20000mg/m3 female animals) after
exposures were initiated. - OK	35

Table 3.

The observation for terminal sacrifice has not been included as an
observation. The observation does occur in Appendix E but has not been
tabulated for the females that were terminated on Day 25 of gestation.
Corrected - OK	Table 3

Table 14, Group 4, Cycle Length.

Please verify the Standard Deviation for this parameter. It appears that
it should be 0.41, not 0.40. Is there another digit that is not shown
that the computer uses in calculations for this parameter? Otherwise, it
is not clear why there would be an error. 	SD rounded to 0.4 - OK	Table
14

Draft Report, Table 16 and Appendix R, Duration of Gestation.

In Table 16 the Standard Deviation is reported to two decimal places
while in Appendix R this value is reported as one decimal place. These
values should agree in both instances.	They do agree but due to computer
program limitations, the results are presented to different numbers of
significant figures.  Appendix R was regenerated with revised rounding
rules (1decimal place) after Table 16 was printed. OK	Table 16

Draft Report, Table 17.

The lacerations for litter 1562 pups 3, 9 and 14 as reported in Appendix
T are not tabulated on Summary Table 17.	Tabulated on Summary Table 17 -
OK	Table 17

Draft Report, Table 18, Page 4, 5 & 6.

These pages report summary data for mean pup weight gain. An individual
data table is not available to support these means. Should an individual
value table be included in the report?	This is not possible due to TASC
computer system limitations.  TASC does the weight gain calculation but
does not generate a table of the individual data. - OK	Table 18

Draft Report, Table 20, Page 5, Skin, Group II, Litter Incidence.

 Pup Incidence should 0, 0 not 1, 1.	OK as is – pup #2571-1F had skin
observations.  - ok	Table 20

Table 23, Appendix Z.

It is unclear from the gross to microscopic pathology data reported in
Appendix Z whether or not the gross lesions were examined for any of the
animals. Only the randomly selected animals should have had gross
lesions examined, but it is unclear for the rest of them. The comment,
“no micropathology observations on tissue” implies that these
tissues were reviewed, yet below it is indicated that, “no tissues
examined”. As well, the summary Table 14 does not tabulate these
tissues as having been examined microscopically. The histopathology
worksheet for animal number 1064 also does not indicate that the lesion
tissue was processed for evaluation. Please clarify the gross to
microscopic appendix to clearly indicate what was examined.	

“no microscopic observations on tissue” and “no tissue examined”
both mean that tissues were not examined by the Pathologist.  If tissues
were examined, then an abnormal observation or “tissue is
unremarkable” would be noted.  OK, I guess.  	Appendix Z

Appendix A, Inhalation Report, Section 2.1.4 Environmental Conditions.

It would be helpful to indicate the frequency and duration of the
excursions from the desired ranges.	No change.  The excursions were
minor and insignificant and a tabulation is not needed   This may or may
not be true.  The reader only has access to the daily mean temperature
and humidity readings.  Excursions could be major and/or significant,
the reader can not tell.  From what the summary data indicates, 50 to
over 70% of the time, the MEAN temperatures were out of the
protocol-specified range, so it would be helpful to indicate further
extent and duration of the deviations.    	Appendix A

Appendix A, Inhalation Report, Table III, page 2.

The last line of the flowmeters indicates P/N D80026B when it should be
D8000268. The last line of the Pressure/Vacuum Gauges should read P/N SG
8383, not S6.  Please verify.	Corrected - OK	Appendix A, Table 3, p. 2

[report p. 44]

Appendix A, Inhalation Report, Table IV, page 1.

For group 2, Starting with the second H-7 sample all were taken on Jan.
12. As well, the H-3 port, in the data is not marked as such, so it is
difficult to differentiate it from the other ports.	Report corrected and
entry for H-3 clarified OK	Appendix A, Table 4, p. 1

[report p. 46]

Appendix A, Table V, Page 1, Miran Calibration.

It is indicated that The following calibration data was used for
exposures or trials between November 15, …… As well, on page 4 of
this section it should indicate that this calibration was used for
exposures between Feb. 12 and March 27th……And then on page 7, it
should indicate that this calibration was used for exposures between
March 28th and termination. It would also be helpful to talk of the
change of Mirans.	Report corrected and clarified - OK	Appendix A, p. 1
[rpt p. 48]

p. 4 [rpt p. 51]

p. 7 [rpt p. 54]

Appendix A, Inhalation Report, CMR Table, Page 12.

Please verify the values for the second and third Miran readings for May
15. It appears they are 2150 and 2330.	Report corrected - OK	Appendix A,
p. 12

Appendix A, Inhalation Report.

Pretest particle size data should be reported.  	Particle size data
added to Table VI in Appendix A  OK	Appendix A. p. 57

Appendix B, Analytical Report, Page 3, Table of Contents.

The title for Table 1 should have the words “/Trials” added after
the word “Pretest” to agree with the table heading.	Report corrected
- OK	Appendix B, p. 3

Appendix B, Analytical Report, Page 3, Table of Contents – The title
for Figure IV should have the “-A.” removed to agree with the Figure
IV heading.	Report corrected  OK	Appendix B, p. 3

Appendix B, Analytical Report, Page 4, Summary.

The first sentence is confusing. More words are needed to explain that
the test substance was obtained from the generated atmosphere of the rat
exposures.	Report corrected  OK	Appendix B, p. 4

Appendix B, Analytical Report, Page 6, Results and Discussion.

The word “pretest” should be added before the word “trials” in
the first sentence to agree with table headings.	Report corrected  OK
Appendix B, p. 6

Appendix B, Analytical Report, Page 6, Results and Discussion, second
paragraph, first line.

Add “(Table 11)” to clarify the table being discussed.  	Report
corrected  OK	Appendix B, p. 6

Appendix B, Analytical Report, Page 6, Results and Discussion.

The calculation used to correct the area % values for other components
should be added. Examples of these calculations should be stated. As
well, there need to be results and discussion he re. Was the test
substance stable and did it provide a consistent exposure atmosphere?
Calculations and examples added to this Appendix B of the report. 
Discussion of the results is in the Results and Discussion section 3.1
of the main report.  OK	Appendix B, p. 6; 

Results 3.1 pp33-34

Appendix B, Analytical Report, Pages 29, 30 & 31.

The trans-2-Pentane, 2 Methylpentane, 2,4 Dimethylpentane and 2,3
Dimethylpentane peaks have not been labeled on the gas chromatogram
figures II, III and IV.	These have not been added but explanatory
footnote added to figures  OK	Appendix B, p. 29, 30, 31

Appendix B, Analytical Report, Page 29, Figure II, peaks for 2,3
Dimethylbutane, N-Hexane and Benzene.

The retention times printed on this 5 figure do not agree with the raw
data values 32.65 vs. 32.780, 39.51 vs. 39.716 and 55.02 vs. 55.556,
respectively.	These are OK as is and explanatory footnote added to
figures.  No explanation of differing retention times given on figures. 
Appendix B, p. 29, 30, 31

Appendix B, Analytical Report, Page 30, Figure III, peaks for N-Pentane,
2,3 Dimethylbutane, N-Hexane and Benzene.

The retention times printed on this figure do not agree with the raw
data values 22.80 vs. 22.947, 32.67 vs.  32.794, 39.52 vs. 39.727, 55.04
vs. 55.577, respectively.	These are OK as is and explanatory footnote
added to figures.  No explanation of differing retention times given on
figures.	Appendix B, p. 29, 30, 31

Appendix B, Analytical Report, Page 31, Figure IV, peaks for 2,3
Dimethylbutane, N-Hexane and Benzene.

The retention times printed on this figure do not agree with the raw
data values 32.67 vs. 32.798, 39.53 vs.  39.736, and 55.06 vs. 55.595,
respectively.	These are OK as is and explanatory footnote added to
figures.  No explanation of differing retention times given on figures. 
	Appendix B, p. 29, 30, 31

Appendix B, Analytical Report, Page 11.

The corrected sample area % for Isopentane Samples 2003 & 3003 should be
reviewed (reported 38.60 vs.  38.34 calculated, 38.62 vs. 38.30).	These
values have been reviewed again and are OK as presented  ok	Appendix B,
p. 11

Appendix D.

The Clinical Observations of Terminal Sacrifice does not appear on the
tables for the male animals. The Appendix D’s observations stop at Day
105 and the summary table (Table 2) summarizes the data to Day 112.
Therefore, the observation for the terminal sacrifice on Day 112 should
appear in Appendix D.	Appendix reprinted inclusive of 107 as last day of
observations.  OK	Appendix D

Draft Report, Appendix N.

The data for Days of Gestation 0-20 has not been tabulated in this
appendix. The summary of this data is presented in Table 12 and this
table summarizes the data for this period 0-20. Therefore, this
individual data should be reported in Appendix N.	Appendix report
corrected  OK	Appendix N

This situation also is present in Appendix O and Table 13. Table 13
summarizes the data for Days 1 to 28 but the individual data is not
reported for this period in Appendix O.	Appendix report corrected  OK
Appendix O

Appendix P, page 1, animal number 1561.

Please verify the Day 49 value for this animal. It was recorded as
“E”, but then the next day it was re-evaluated and changed to
“D” with no apparent reason given.	Correct stage is D and reason
given in edit tracking print-out. [Note Appendix P was regenerated to
reflect the day on which estrus cycles were monitored within the
exposure period.  Estrus cycle monitoring started on test day 49 and
continued for 21 days before mating was started]  OK	

Appendix P

Appendix P, page 2, animal number 2557.

Please verify the day 49 value for this animal as well. It is indicated
“E” in the data.	Correct stage is D and reason given in edit
tracking print-out.  OK	Appendix P

Appendix R, page 1, animal number 1567.

Please verify the duration of gestation for this animal. It appears to
be 21 days. (All other animals’ duration of gestation is based on the
day delivery begins).	Gestation was from 3 April to 25 April which is 22
days as reported  OK	Appendix R, p. 1

Draft Report, Appendix T.

The observation of “found dead” for Litter 2575 Pup 1 has not been
reported in this appendix. The pup was found dead on Day 0.	Pup was
found dead during delivery and therefore a Day 0 observation was not
recorded.  A preface page with explanation has been added to this
appendix  OK	Appendix T, added new p. 1

Draft Report, Appendix Y, Preface.

Table of Contents states P0 information.  This information should be F1
information. Appendix Y is the Pup Organ Weight data, not P0 data.
Report corrected OK	Appendix Y, preface p. 1

Appendix AA, Sperm Analysis Report.

It is unclear how the Standard Deviations were rounded in the Summary
Table 1. The Testicular Count for the Group I, Standard Deviation is
16.35 (not rounded), yet the Motility Standard Deviation of 4 is rounded
from 3.49. Please clarify.	Standard deviations rounded to same number of
decimal places as mean values OK	Appendix AA, Table 1 (p.10)

Raw Data Issues

Raw Data, Group III, Females, Animal #3552.

A terminal sacrifice printout with final body weight was not available
in the maternal data notebook. A necropsy printout was present in the
fetal pathology notebook.	Weight was not collected and a protocol
deviation was added to the data and report.  OK

	Raw Data for Female 1557.

A printout of the pathology data was not present in the Fetal Pathology
Notebook. The total implantations (13) could not be verified.	Data has
been reprinted and added to study file.  OK

	Raw data for Animal 1557, Female, Group I.

A necropsy printout was unavailable for the necropsy observations. All
other females have printouts that state the findings or state “no
gross lesions”. 	Data has been reprinted and added to study file.  OK

	All chamber trial data and memos explaining what happened when the
Ethanol and Tame studies were switched needs to be included in the data
for this study. When study numbers change, all documentation justifying
that change must be kept with study records. As well, all data needs to
be checked to verify that correct study numbers are present on all data.
Explanatory memo was added to data and study numbers corrected and
footnoted as needed.  OK

	Test material inventory records.

For large tank #10, the use of 8311.2 g needs to be added to the
tank’s inventory.	Added.  OK

	Test material receipt records.

It was not clear whether large tank #12 was also received on 4/25/01.
Please clarify and add any other dates to the report, if appropriate.
Tank #12 was also received on 25April 01 and data clarified.  OK

	Test material inventory.

Tank #10 has no net weight due to an error. If this weight is usually
taken from the Chevron value, why was it not added?	Tank #10 has net
weight of 520.0 pounds per fiber tag on cylinder as per my email of
25April01 to Pharmacy.  OK

	Analytical data.

There is a memo dated 7/30/01 concerning the change from split vs.
splitless injections. This memo is copied and the date of 03 July 01
appears on the copy. Please clarify that this was dated in error.	Date
was corrected.  OK

	Raw data for Animal 3558, Male, Pup 1.

Data records the observation as “no observed pup necropsy finding”.
Edit documentation for the thymus finding of “discolored slight, left,
tissue saved was not present in data”. An edit trail should be
available for this change.	Data was reviewed and the edit trail is
intact.  OK

	Raw Data.

A note to the data should be written to clarify that the Maternal Body
Weight, Clinical Observations and/or Body Weight, Clinical Observations
and/or Food Fed or Food Left values can be located in either the
maternal or litter data notebooks. Both sets of notebooks need to be
reviewed to fully track the maternal data.	A note was added to the Data
Review page to clarify.  OK

	

211(b) Toxicology Research Group

ETOH One-Generation Reproduction Toxicity Study Report 

Reviewer Checklist

  PAGE  1 	  DATE \@ "M/d/yyyy"  11/6/2007  Final