Document ID: EPA-HQ-OAR-2003-0065-0477
Agency: epa
Document Type: Supporting & Related Material
Title: 
Posted Date: 2008-08-04T04:00Z

Comments	Action/Response	Page #

EPA 

ORD finds that., in general, the six developmental toxicity studies were
conducted according to the EPA test guidelines. At this time, however,
we cannot recommend approval of the study reports as submitted, because
we have identified several deficiencies in the statistical analyses and
reporting of data. The attached memoranda provide specific comments on
each of the study reports.	No action required

	Additionally, in our review of the developmental toxicity study
reports, we noticed that certain data on dams that delivered early were
omitted from the appendices because the data were excluded from the
statistical analyses. Please note that all of the data generated as part
of the21 l(b) testing program must be provided to EPA for review, even
if they are negative or are not used in the statistical analyses.	Data
added to report	C-4, C-8, AND D-3

We strongly encourage the RG to address our comments before revised
versions of the reports are submitted to EPA. The RG should also
adequately respond to the comments raised byte independent peer
reviewers and provide the additional information requested. To help our
reviewers complete the reviews of the revised study reports, we also
request that a summary of the changes or marked-up copies be provided.
Checklist prepared

	In general the NHEERL reviewers concluded that the studies were
conducted in accordance with the testing guidelines; however, several
issues were raised that warrant further attention. Although not
essential, it would be preferable to include tables into the text that
accurately summarize developmental toxicity data, showing some of the
key endpoints, e.g., corpora lutea, implantations, live fetuses,
preimplantation loss, post implantation loss, and total malformations,
variations, and affected implants (from pages F-1 and F-2).	All tables
removed from text at Sponsor’s request

	The study authors concluded that the maternal body weight data did not
indicate maternal toxicity, and regarded the significant decrease in
weight gain on GD 20-21 to be an apparent spurious effect. However, the
report text failed to mention that, according to page C-2, there were
also significant linear responses for reduced weight gains on GD 8- 1 1,
5-2 1, and 0-2 1 as well as for the extrauterine weight gain on GD 0-21.
Furthermore, the high-exposure group showed consistent, albeit
nonsignificant, reduced weight gains at all other intervals examined.
Collectively, we regard these weight gain data to be evidence of slight
maternal toxicity at 20,000 mg/m3.	Revised abstract, summary, results,
and discussion	i, I-1, I-2, 4-1, 4-2, 4-5

The presentation of data in Tables 1-1 and 4- 1 show the N value as the
number of fetuses, implying that the fetus, not the litter, is being
used (inappropriately) as the experimental unit of analysis. This
coinflicts with the summary table on page G-1, where N is the number of
litters; the means and SD values are identical in all three tables. The
Methods indicate that the analysis was appropriate in that a nested
design was used (fetal weights were nested in litters). To reduce
concenns of inappropriate statistics, the Tables 1 - 1,4- 1, and on page
G- 1 should indicate that a nested analysis was used. The number of
litters as well as fetuses should be shown be shown in all three tables.
Tables 1-1 and 4-1 removed.  Table G-1  revised to reflect the revised
statistical analyses	1-1, 4-2, G-1

The statistical analyses of the fetal examination data are inappropriate
and should be revised. In the analyses (chi-square and Fisher's exact
test), the study authors used the fetus, rather than the litter, as the
experimental unit. Since fetuses from the same litter are not
independent of each other, a fetus-based analysis inappropriately
inflates the degrees of freedom. (The study authors also did chi-square
and Fisher's exact test on litter incidences; this is statistically
valid, but is inadequate without additional statistics because it
doesn't consider within-litter incidences and can lead to false
negatives.)	Data reanalyzed	3-11, Appendix K

For dams that delivered early, the individual body weight and food
consumption data are omitted from Appendices C and D. It is acceptable
for these animals to remain excluded from statistical analyses. However,
because treatment could affect gestation length and premature delivery,
it is important that the data from these animals still be included in
the appendices and available for review.	Data added to report	C-4, C-8,
AND D-3

Peer Reviewer – Schlesinger

Overall, 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 conclusions as presented are sound.	No action required

	There are some concerns about a number of statistical issues that have
been previously raised in regards to related reports. These are
indicated below.	Data reanalyzed

	Specific comments:

Exposure Schedule:

It is stated that the exposure period was "at least" 6 hours per day.
What was the range of actual daily exposure durations.	Revised to six
hours	3-7

Page 3-8. Statistical Analysis:

When the Bartlett's test indicated nonhomogeneity of variance the
investigators used a nonparametric analysis of variance. An alternative
approach would be to use some transformation that would have resulted in
variance homogeneity, and then a parametric analysis of variance could
have been used. This apparently has been used for some of the analyses
are reported. This uniformity would have made the statistical analysis
of all data sets much more internally consistent and would not present
any potential for Merent types of tests having different degrees of
conservativeness in detecting an significant changes from air control.
In any case, the investigators should indicate which specific
statistical tests were used for which datasets.	No change; the footnotes
with the mean data indicate which tests were performed

	The investigators also indicate that they performed statistical testing
with both transformed and nontransformed percentage data but that the
former where not reported since they were not statistically significant.
This implies that whichever procedure resulted in significance would be
reported.  This is an improper use of statistical techniques. If data
need to be transformed because they are not normally distributed as raw
data then the statistical tests with these transformed data are the ones
that should be reported. There cannot be a "picking and choosing" of
data sets to report.	Results of the transformed data added to the report
F-1, F-2

It is also stated that statistical tests were conducted at both the 5%
and 1% significance levels. The justification for this is not apparent.
One of the a priori decisions that should to be made before the study is
conducted involve 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.	Clarified in the text
3-10

Page 4-1 Gestation Body Weight:

The Appendix L is initially confusing, since each summary table is not
individually labeled as to whether it is actual weight were in the body
of the main text rather than in the appendix. or weight change.
Furthermore, it would be better if all of the summary tables 	Appendix B
revised to clarify data types presented.

No change in the location of the summary tables	Appendix B

Table 4-2. There does seem to be somewhat of an exposure level-related
increase in stunted growth, although the numbers are indeed small.	Table
4-2 removed from report	4-2

Page 4-6. Exposure Data:

It is stated that the particle levels were 1 and 1.5 mg/m3 and that this
represents a “minimal” aerosol component.  Firstly, what was the
composition of the aerosol; were there HEPA filters on the exposure
units?  Secondly, in the opinion of this reviewer, these reported levels
of particles are not minimal but are high for a system in which there
should be total vapor.	No change.  The source of the dust is the make-up
air from the animal room which was not filtered.

	Page 4-7. Discussion:

There clearly were changes in the skeletal variations at the mid level
of exposure. While there was no exposure concentration related pattern,
the investigators should provide some possible explanation as to why
these changes may have occurred before discounting them in the
determination of the no effect level.	No change

	Appendix H (p. H-1):

The statistical notations on this table are not clear. For example, the
total fetuses with skeletal abnormalities seem to be significant with
the chi square test and the Fisher test. This needs some clarification.
Statistical notations removed from Appendix H.  Statistical details are
in Appendix K	H-1 – H-5 and Appendix K

Peer Reviewer – Goldsworthy

It is interesting that gasoline with MTBE, but not BGVC only, also noted
a high incidence of bifid centra of the thoracic vertebrae. Historical
control data range  presentation for these findings should be noted if
possible. 	Historical control data presented in the Discussion	4-5

I agree with the report's conclusion that these decreases in the present
study are likely not to be treatment related or biologically
significant. These rare findings may need to be further assessed in the
context of the entire database API is generating in this testing
initiative.	No action required

	Results section should present (in table format) the total skeletal
variations data and total visceral variations data, especially in light
of the significant increases in the 10,000 mg/m3 group for total
skeletal variations.	No change

	211 (b) Research Group Reviewer 

General Comments

The final report should have sequential numbering in addition to the
A-1, A-2, etc. system so that we can be certain that we have all report
pages.	Sequential numbering added

	There is no Appendix K, Historical Control Data, included with the
report. This should be reviewed prior to report finalization.	Historical
control data added	Appendix K

The QA Statement is blank. Before the report is finalized, we should
receive a revised QA statement that lists all audited phases of the
study and the audit dates. The report should not be finalized without
sponsor review of the QA statement.	QA statement added	ix

Tables with nested analysis (fetal data) need to list the number of
litters per exposure level.	Number of litters presented	G-1

The statistical programs include linear trend analysis but no criteria
for ignoring statistically significant linear regression, which appears
to be the procedure in place when the intergroup comparisons is not
statistically significant. There is also a test for linear lack of fit,
but it isn't clear if this is routinely performed or only when the
intergroup comparisons are negative, or some other rationale. The
decision tree for the statistical programs needs to be explained more
fully, and the criteria for dismissing statistically significant
findings should be described in more detail also.	Linear trend analyses
results added to the results	4-1, 4-2

There are a number of statistically significant linear regression
analyses for gestation body weight change and food consumption that
could be considered justification for a alternate interpretations of
maternal data. These are not addressed in the text, which I believe will
need to be done to appropriately justify the data interpretation.
Abstract, summary, results, and discussion revised	i, I-1, I-2, 4-1,
4-2, 4-5

Specific Comments (by page and section)

Page ii, Table of Contents:

Justification of Dosing Route and Justification of Dose Selection both
appear on page 2-2, not 2-1.	Corrected	Ii

Page iv, Table of Contents:

Appendix K is missing.	Appendix K added	V

Page viii, QA Statement:

As noted above, this should list the phases and dates of inspection.	QA
statement added	ix

Page 1-1, Summary:

First paragraph, Lines 6-9: There should be more of a connection between
"The Sponsor selected the doses." and the next lines. Something like
"based upon the following considerations" would be helpful. Also, there
is no justification provided for why 2K was expected to be a NOAEL.
Comment regarding the overall testing program might be appropriate.
Revised to the statement supplied by the Sponsor	1-1

Third paragraph, line 5: Cesarian should be spelled out, as in the next
paragraph.	Revised	1-1

Page 1-2, Summary:

There is no reason to include a table of fetal weights in the summary
when this is not a parameter that is interpreted as being affected by
exposure, and there are no unusual findings to clarify. This table
should be deleted.	Table removed	1-1

First paragraph, lines 1-2:

The sentence should be: "...and fetal growth resulted& no signs...".
Revised	1-2

First paragraph, lines 3-6:

There's no need to repeat the information presented on the previous page
regarding bifid thoracic vertebral centra. The last two sentences in
this paragraph should be deleted. The final sentence of the summary,
regarding the NOAEL, could be moved into the same paragraph as the "In
conclusion" sentence.	Removed	1-2

Page 2-1, Introduction:

First paragraph, first sentence: "...administered& whole-body...".
Revised	2-1

Justification for Selection of Test System:

I believe the reference should be 1994 (for the 21 1 b program), not
1996 (the testing guideline).	Reference corrected	2-1

Page 2-2, Justification of Dose Selection:

Adequate justification for dose selection would provide the reasoning
for why 2K was expected to produce no effects. Since the mid-dose was
probably not expected to cause effects, it wasn't selected to produce a
dose response.	Added the statement supplied by the Sponsor	2-2

Page 3-2, Characterization of the Test Substance:

All of this information as described in the first paragraph should be
included in the report as an appendix. It (MRD-00-714?) could be
included withhear and compared to the data in Appendix J.	No change. 
There is a separate report for this data

	Page 3-3, Analytical Concentration:

Line 5: "...analyses showed major components of the test atmosphere and
were used to assess ..."	Revised	3-3

Page 3-3, Particle size analysis:

Line 2: There should be a space between "the" and "control"	Added	3-3

Page 3-4, Feed and Water:

Both the feed and water analyses should be included in the report as
appendices.	Analyses added in Appendix M	Appendix M

Page 3-6, Administration of Test Substance and Exposure Schedule:

Line 1: There should be a space between "M3" and "inhalation."	Added	3-7

Page 3-7, Euthanasia:

The method of euthanasia for viable fetuses must be included.	Added	3-8

Page 3-8, Tissue Preservation:

The method of storage/preservation for the skeletal specimens needs to
be included also.	Added	3-8

Page 3-8, Statistical Analysis:

Second paragraph: Transformations need to be reported when performed,
whether statistically significant or not.	Transformations reported	F-2

Third paragraph: The lab needs to provide explanations for: (a) why
linear regression is performed when there isn't a difference between
groups; and (b) what criteria is used to ignore statistically
significant linear trend performed in such situations. What was the
criteria for performing a lack of fit test?	Linear regression and the
lack of fit test run automatically with the statistical analyses.  The
results have been reported.

	4-1

Page 3-9, Statistical Analysis, continued:

Second paragraph: Was Armitage's test for linear trend always performed,
or only if the Fisher exact was statistically significant?	No longer
applicable

	Page 4-1, Clinical lnlife Observations:

Second paragraph: Please revise the third sentence to indicate that red
ocular discharge and dry red ocular discharge were observed in the same
female.	Added	4-1

Page 4-1, Gestation Body Weight:

The statistical significances (linear regression) for GD 8-1 1, 5-21,
0-21, and 0-21 C need to be addressed. The data for these intervals
suggest a slight effect upon the 20K group, which is not considered
biologically significant by the authors when statistically significant
for the GD 20-21 interval; therefore it is appropriate that the authors
note the reason(s) why the signficant regressions should not be
considered biologically important.	Linear regression results added to
the results.	4-1

Page 4-1, Gestation Food Consumption:

The statistical significances (linear regression) for GD 5-8,8-11, 11
-14, 20-1 1, and 5-20 need to be addressed. As with body weight change,
the data suggest a slight effect upon the 20K group. Since this has not
been considered biologically significant by the authors, there should be
some justification provided here.	Linear regression results added to the
results.	4-2

Page 4-2, Gross Postmortem Observations:

"Dark red material" should be identified by location. This observatation
and the observations of "placentas," "dark red material in the uterus,"
"dark brown material in the stomach and intestines," and "fetus in the
cervix" do not appear as these descriptions on the summation of
observations on page E-1.	Added to results and Appendix	4-2, E-1

Page 4-2, Uterine Implantation Data:

Referring to page F-2, total variations: Why was a "lack of fit" test
performed for the linear response, when the linear regression was not
significant?	Lack of fit test runs automatically.  In this case it
indicates that the linear gression was not an appropriate regression
model for the data.

	Page 4-2, Fetal Body Weight:

Table 4-1 should be deleted; it repeats information presented in
Appendix G and is unnecessary for study interpretation.	Table 4-1
deleted	4-2

Page 4-3, Fetal Observations:

Table 4-2 should be deleted; it repeats information presented in
Appendix H and is unnecessary for study interpretation.	Table 4-3
deleted	4-3

Page 4-4, Visceral Observations:

It isn't clear if visceral observations are considered to be
developmental toxicity findings, artifactual, or something else. This
needs some clarification.	Clarified	4-3

"Abnormal abdominal contents" is not an informative description.	Revised
4-3

Table 4-3 should be deleted; it repeats information presented in
Appendix H and is unnecessary for study interpretation.	Table 4-3
deleted	4-3

Page 4-5, Skeletal Observations:

Second sentence: There is no explanation (in this section) of the
reasoning for why the statistically elevated total skeletal variations
and bifid thoracic vertebral centra are not considered effects related
to exposure. Please include (noted in summary).	Explanation added	4-3

Table 4-4 should be deleted; it repeats information presented in
Appendix H and is unnecessary for study interpretation.	Table 4-4
deleted	4-3

My count for bifid thoracic centra for the 10K.group was 34 rather than
33, and for 20K was 17 rather than 16. Please check these numbers.
Numbers checked twice.  Replaced 16 with 17 for 20K	H-4

Page 4-6, Exposure Data:

Appendix J is not referred to in the report; shouldn't it be referred to
in this section?	Added	4-4

Final paragraph, final sentence: room^"	Added the “s”	4-4

Page 4-7, Discussion:

Third paragraph, second and third paragraphs: Please delete these
sentences, since they repeat information provided in the previous
paragraph.	Deleted	4-5

Page 4-8, Protocol Exceptions:

Chamber temperature and humidity: "protocol-defined	Corrected	4-6

The temperatures and humidity values outside of the protocol-defined
range should be provided here, as well as a summation sentence that
provides the range of values.	The range of values is presented earlier
in the report.  The section was revised to reference Appendix I	4-6,
I-23 – I 30

Page 5-2, Fetal External and Visceral Examinations:

Observations are defined as not malformations and not variations, but
not defined by whatlhow they differ from normal, nor if they are
considered to be developmental toxicity endpoints. Please clarify.
Revised section	4-8

Appendices:

Appendix C:

Page C-1, C-2: Delete "Run 1 ."	Deleted	C-1, C-2

Appendix D:

Page D-1 : Delete "Run 1 ."	Deleted	D-1

Appendix E:

Page E-1: The descriptions summarized here are not consistently
described as noted on the individual observations and/or in the text on
page 4-2.	Revised	E-1

The observations associated with the early delivery should be noted as
such and probably reported in separate rows of the table.	Revised	E-1

Appendix F:

F-2, total variations: Why was a "lack of fit" test performed for the
linear response, when the linear regression was not significant?	No
change. The lack of fit test runs with every analyses

	Appendix G:

The group sizes based upon the number of litters needs to be presented.
The individual data need to present the mean male and mean female fetal
weights for each litter.	Number of litters presented	G-1

Appendix H:

Page H-2: "Abnormal abdominal contents:" what is this?	Revised	H-2

Page H-4: My count for bifid thoracic centra for the 1 OK group was 34
rather than 33, and for 20K was 17 rather than 16. Please check these
numbers.	Numbers checked twice.  Rvised 16 to 17	H-4

Pages H-26, H-28, H-30, H-37, H-39, H-47, H-48, H-53, H-64, H-71, H-74,
H-87: Please provide the reason(s) that some fetal specimens needed to
be arbitrarily assigned fetal numbers.	Reason added	H-26, H-28, H-30,
H-37, H-39, H-47, H-48, H-53, H-64, H-71, H-74, H-87

211 (b) Research Group QA/QC Reviewer

The following items require further consideration:

Page i, Abstract and throughout the report:

when indicating concentrations of test article, indicate target
concentrations (unless they are the actual ones measured).	Target added
Throughout the report

Page vii, Compliance Statement:

The sponsor also needs to sign a compliance statement. It can be a
separate one from the Testing Facility’s, but there must be one signed
by the sponsor.	Space added on com0pliance statement for Sponsor’s
signature	vii

Compliance Statement:

Since it was 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.	Added to the test substance section of the report.  We do not
consider this to be a compliance issue as it is now available.	3-1

Page viii, Personnel:

Is a compound preparation supervisor an appropriate person to have
listed under personnel? Did this person actually work on this study?	No
change.  Required by protocol

	Page ix, QA Statement:

The QA statement needs to be completed.	QA statement added	ix

Page 2-1, Experimental Date:

This date should be March 21 or later as the skeletal re-evaluation was
done on March 21 (Experimental termination should be the last date that
data are collected)	Date revised	2-1

Page 3-1:

During the audit, no real “test material receipt record” could be
found. It appears that dispensing received the material on 4/10/01 and
on 6/14/01, but none was available for when the test material arrived at
EMBSI. All records should be searched to find the original receipt
record to determine the exact date on which this test material was
received at EMBSI.	The test substance was logged into our data and this
is the documentation of receipt.  

	Page 3-1, The container numbers:

The container numbers for the 6/14/01 receipt date are confused.
Container 9A is correct, but the next container should be 9B(2b, large
container) as should containers 10A, 11A and 12 A were all dispensed
from large container 2. Please clarify.	Section revised	3-1

Data sheets for containers 9A-12A should indicate manufacturer’s
number 2, the large tank number.	Added to data

	Page 3-1, Materials and Methods, Test Material:

The expiration dates given are for five years. Are there data to support
these expiration dates? It should be kept with study data.	Expiration
dates were not supplied by the Sponsor.  The expiration dates used were
per EMBSI SOP

	Page 3-2, Test Material, Analytical Concentration:

It is indicated that chromatographic analyses showed major components of
the test atmosphere and was used to assess the stability of the test
substance over the duration of the study. The number of components and
their identities should probably be indicated for reproducibility
purposes.	Added the number of components.  	3-2

The next paragraph should indicate that this analysis was done to
determine component proportions of the test material atmosphere compared
to the liquid test material.	Added	3-2

Page 3-5, Environmental Conditions:

These are the protocol-required ranges for temperature and humidity. The
actual measured ranges should be given.	The actual measured ranged were
added for the chambers.  The animals rooms were monitored to determine
that they were within the acceptable range, but values were not
recorded.	3-5

Page 3-7, Test Atmosphere:

The daily mean exposure concentrations were “intended” to be within
+10% of the target exposure levels.	Revised statement to include the
protocol deviation	3-8

Page 4-1, Gestation Body Weight:

One mid-dose and one high-dose animal lost between 22 and 27 grams
between GD 20 and 21. This may have affected the means at this interval,
particularly the high dose group.	Body weight data and food consumption
data for GD 21 and other affedted intervals not used for the statistical
analyses for IGK757 (high dose animal).  This data was excluded because
the animal was attempting to deliver its litter on GD 21.	i, I-1, I-2,
4-1, 4-2, 4-5, C-1, C-2, C-6, C-10, D-1, D-5

Page 4-2, Gross Postmortem Observations, first sentence:

The dark red material and placentas were in the “stomach of the”
2000 mg/m3 dam…….	Revised	4-2

Page 4-4, Table 4-3:

Aneurysm is misspelled.	Table 4-3 deleted	4-2

Page 4-6, last sentence:

The light intensity in the chamber room ranged from 4.6 to 40.0
footcandles.	The light intensity in the chamber room was 32 to 40
foot-candles.  The low range for the animal room was 4.6 foot-candles
4-3

Page 4-12, Protocol Exceptions:

Having no analytical data (hydrocarbon distribution) for the 20,000
mg/m3 group for the week 3 interval should be included as a protocol
deviation. This was required data.	Added	4-6

Page 4-12, Protocol Exceptions:

The extent of the temperature and humidity excursions needs to be given.
Some sense of how frequently, for how long, and what temperature and
humidity were reached should appear.	Section revised to refer reader to
Appendix I where all excursions are identified	4-6

Page C-1:

The standard deviation for the GD 11 weight for the 10,000 mg/m3 group
should be 19.66. Shouldn’t this round to 19.7?	We calculate 19.55
which rounds to 19.6.  No change

	Page C-8:

The column heading next to the GD 20-21 should indicate GD 5-21 not 0-5.
Corrected	C-8

Page E-1:

There is an entry of Cervix, abnormal contents. The one animal in the
high dose that had a finding in the cervix, had a fetus in the cervix,
not really abnormal contents.	Added that a fetus was the abnormal
content	E-1

Page E-4, animal number 741:

It should probably be noted that 17 live pups were delivered.	Added	E-4

Page F-2:

It is not clear why there are only 21 litters considered in the low-dose
group for % Preimplantation Loss. The mean using 22 liters is 2.4.
Corrected	F-2

Page F-2, % Postimplantation Loss, Low Dose:

It is unclear where the value of 6.3 comes from. The audit found the
mean to be 2.6+4.3. Please verify.	Corrected	F-2

Page G-2, the first fetus in each litter is only report to one decimal
place where all others have two. It appears that the column is just
missing the digit, as they are not rounded.	Corrected	G-2

Page H-4, please verify the incidence of “Vertebrae, Thoracic Centra
Bifid,” as it appears there are 21, 16, 34, and 17 affected in the
control, low, mid, and high dose, respectively. Please verify and change
corresponding tables within the text if necessary.	Data verified twice. 
Values are 21, 16, 33, and 17.	H-4

Page I-4, Particle size, second paragraph:

A particle size determination ……. from the control and 20,000 mg/m3
concentration.	Revised sentence	I-4

Page I-4, Particle size, second paragraph. Is there any way to make the
explanation of the particle size correspond to the data reported? Can it
be simplified at all?	Second paragraph revised.  Added a third paragraph
I-4

Page I-10:

There are two GC calibrations labeled with the date, 10/31/01. Please
clarify in the data, which one is used and verify that the one reported
is the correct one.	Clarified in data and reported calibration verified
I-9

Page I-11, 20,000 mg/m3 data:

The mean concentration minimum should be 19,913 mg/m3. The nominal for
the last day, should be 19,486. This changes the mean to 20,127 with a
standard deviation of 479. Please verify these changes .	Corrected

The nominal for the last day is 19556 as reported.  No change	I-10

Appendix J:

Several pages are labeled as J-1.	Pagination corrected	Appendix J

Appendix J, Page J-1:

The procedure for collecting the charcoal tubes needs to be described
somewhere in the report (either in Appendix I or J). A description of
how they were stored prior to receipt in the Analytical Chemistry Lab is
needed as well.	Collection and storage procedure added to the Test
Substance section of the report.

	3-2

Appendix J, Page J-1, Sample Preparation, next to last sentence:

charcoal tube sections were ………..for at least 60 minutes according
to the data.	Corrected	J-1

Page J-1, Characterization:

MTBE in the first sentence should be ETOH.	Corrected	J-1

Table J-1:

Data from the analysis of the neat material would be helpful in showing
that the test material is stable in vapor phase.	No change.  This is in
the characterization report

	Page J-2, Results:

There should be some indication of what the data show. There was no
change in test material composition for the duration of the test, at
different dose levels, etc.	Added to the last sentence of the first
paragraph	J-2

Table J-1:

For the Dec. 3, 2001 sample in the 2000 mg/m3 group, the n-butane value
should be 12.2. Please verify.	Corrected	J-3

Table J-1:

The footnote indicating that there was no breakthrough in any of the
samples except sample 9 is not true. There was breakthrough apparently
for sample number 7. It appears from the data that this sample was
repeated and no breakthrough was seen in the repeat sample. There are
two problems with this. First, it is unclear how a repeat sample could
have been run, as all of the samples were collected and stored until
analysis. No repeat sampling could have occurred by the time the
breakthrough was noted.

Second, the data in Table J-1 should be footnoted as a repeat sample
with an explanation (if this was indeed the case).	The statement is
true.  The apparent breakthrough on sample number 7 was carryover in the
column from the previous analysis.  The sample was re-injected and the
results reported.  A footnote was added to the data and Table J-1	J-3

Historical Control:

No historical control data are reported. These data need to be reported.
Historical control data added	Appendix L

Data for the animals that delivered early or those that were not
pregnant need to be reported somewhere in the report. A separate table
for those animals needs to be created.	Data for animals that delivered
early were added to Appendices C and D	C-4, C-8, AND D-3

Data Issues:

Animal number 800 had 18 Corpora Lutea and only 14 implantation sites.
This should have been verified (per SOP) by an independent technician,
but was not. This would be considered an SOP deviation and should be
documented as such.	Documented and acknowledged by the Study Director as
SOP deviation

	Analytical data:

The GC print-out for Analytical Reference 171434-001B should have been
171434-002. The rest of the samples were also designated incorrectly
(001-C should have been 003, 002A should have been 004, etc.)	The
printouts are designated correctly.  No change

	All chamber trial data (GC print-outs) indicate study number 169534R
and the incorrect test material number. Please verify and correct.
Corrected

	The data trail from collection of the charcoal tube samples in the
inhalation chambers to the freezer before being transferred to the
analytical lab is not clear. There is no documentation to verify that
these samples were taken and frozen until submission to the analytical
lab.	Memo added to data describing how samples were stored.

	Since charcoal tube samples from chambers were apparently frozen until
all samples could be analyzed, there needs to be a stability study done
to verify that these samples are stable in the freezer for this period
of time.	No change.  This is the standard method for storing volatile
samples on charcoal tubes.  Once the substance is on the charcoal, only
high heat or a solvent will remove it.  Additionally, the data reported 
in Appendix J shows that the samples are stable.

	There needs to be a clear indication of what the analytical lab
considers raw data. When data are not printed out until a week after the
analysis, it is not appropriate to consider the paper print-out to be
data. Since the GC program is storing data, the computer system must be
completely validated and follow all of the requirements of an on-line
data collection system (including change-control procedures, limited
access, complete maintenance of a data trail, etc.). Please verify that
this is the case.	The printout is the raw data.  We have tested the
security of the chromatogram in the system and it is secure.  All other
operations around the chromatogram are documented.

	There is a notation in the room log that extra animals from this study
were moved from PE103 to 104 on Dec. 3. There is another notation,
however, that extra animals from PE105 (Study 117150C) were moved to
PE103 on Dec. 7. Please clarify and verify that another study’s
animals were not in this study’s animal room.	No change.  These were
untreated animals of the same health profile as the Study 171434
animals.

	Rack use/change log:

There are no study numbers on these sheets beginning on Nov. 6. Please
clarify.	Receipt date was recorded, which was acceptable.

	Skeletal evaluation data:

The late entry of NOA for those animals that were re-evaluated was not
always error-coded. Please correct.	Corrected

	Test material use log:

It appears that there was a sizeable amount of test material
(approximately 1,135g) used between chamber trials and the first
exposure that is not accounted for. Please explain.	Memo added to the
data.  It was used for sorbent tube sampling method development

	All of the Analytical Method Validation data (from before the samples
were run and after the samples were run) need to be compiled in a report
to show which solvents were best at extracting ETOH and that the 10%
2-propanol did not interfere with the other components during the
analyses (It is assumed that that is the reason for analyzing the 10%
2-propanol and test material samples after the study was completed).	No
change.  This activity is part of the characterization study.

	It appears that the only identifier on the individual GC print-outs on
the daily chamber analyses is “Inhalation Staff”. The responsible
technician has initialed the cover sheet, but on the actual data
print-out, this person is not identified. Now that more than one
technician runs the inhalation exposures, the initials of the
responsible technician should appear on the individual data print-outs.
Memo added to data

	The GC print-out of the butane standard checks are not identified as
such. Please label these data with appropriate identification.	Memo
added to the data.  All butane standard checks are in their own section
of the data.

	Analytical Sampling Record in the Inhalation Lab only includes study
intervals 11/27, 12/3,12/10,12/17 in order of collection. The method
development samples were recorded out of order. Please explain.	The
method development samples were separated from the study samples to make
the data less confusing.

	

211(b) Toxicology Research Group

Gas+ETOH Rat Developmental Toxicity Study Report 

Reviewer Checklist

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