Document ID: EPA-HQ-OAR-2003-0065-0480
Agency: epa
Document Type: Supporting & Related Material
Title: 
Posted Date: 2008-08-05T04:00Z

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 change 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 the 21 l(b) testing program must be provided to EPA for review, even
if they are negative or are not used in the statistical analyses.	No
dams delivered early in this study.  No change

	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 by the 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 completed for changes that were made.

	In general, the API response to the independent peer reviewers'
comments appears to adequately address the reviewers' comments; however,
the study report available for review has not yet been revised in
accordance with the API response. In addition, we offer input regarding
one of the issues raised by one the i.ndependent peer reviewers and the
API response:	No action required

	

The current report, unlike others, contains no summary tables of
maternal or developmental data. The API response indicates a preference
that tables presented within the report text be only those tables
:necessary to support "additional interpretive analysis" not clearly
provided by the accompanying appendices. We strongly disagree. Although
not absolutely essential, we highly recornmer~dth at reports routinely
contain summary tables of key developmental toxicity data (e.g., corpora
lutea, implantations, live fetuses, preimplantation loss,
postimplantation loss, fetal weights, and selected fetal examination
findings), even when the data are negative. As alluded to by Dr.
Goldsworthy, such tables substantiate the reported findings (both
positive and negative) in a more cogent manner than the appendices, and
greatly enhance the review process.	No tables added to the report text

	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.

The report text did not mention that, in addition to the significant
effect on maternal weight gain on GD 8-1 1 :, there were also
statistically significant linear dose responses for weight gain on GD
5-8 and 1 1-14 as well as for extrauterine maternal weight gain on GD
0-21. These data are further evidence of a treatment effect at the
highest concentration.

Given the data for extrauterine weight gain on GD 0-2 1, there appears
to be a lasting effect of treatment; thus, it is questionable to
describe the maternal toxicity evidenced here as "transient".	Linear
trend results added to results.

Transient removed from the abstract and summary	4-1

i, 1-1, and 1-2



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 witho-ut additional statistics because it
doesn't consider within-litter incidences and can lead to false
negatives.)	Observation data reanalyzed as discussed with EPA	Appendix K

Analyses of fetal examination data should combine incidences of related
findings to better show trends that may be treatment related. Examples
of related findings that could be combined include:

vertebral centra variations (including thoracic and lumbar; including
unossified, bifid, and dumbbell-shaped)

supernumlerary thoracic/lumbar ribs (including rudimentary and
well-formed ribs on Ll and T 14)	Observations combined as necessary for
the reanalyses	Appendix K



The statistically significant increased fetal incidence of unossified
sternebra #5 was not regarded as evidence of developmental toxicity by
the study authors because the litter incidence was not statistically
significant.

True, the litter is the appropriate unit of analysis; however, as stated
above, the litter analyses (chi-square and Fisher' exact) presented in
the report do not consider withinlitter incidences and can lead to false
negative results. Thus, pending re-analysis, we tentatively regard the
increased incidence of unossified sternebra #5 to reflect developmental
toxicity at 20,000 mg/m3.

The study authors point out that sternebra #5 is the last to ossify and
that it's appearance is the most variable. This is hardly a reason to
dismiss the finding; to the contrary, it is this normal variability that
makes it conducive to detecting treatment-induced delays in
ossification.

Although sternebral ossification provides an index of developmental
delay, other ossification sites (e.g., caudal vertebrae, metacarpals,
metatarsals, phalanges) also show normal variability and, when
quantified, often are more sensitive in detecting developm.enta1 delays.
Apparently, however, the skeletal examinations did not attempt to
quantify the extent of ossification at such sites. In view of the data
suggestive of a treatment,-induced delay in sternebral ossification, we
recommend that the study authors evaluate the number of ossification
sites at one or more of these sites (e.g., the number of ossified caudal
vertebrae).	The revised statistical analyses did not detect any
statistically significant differences between the control group and the
groups exposed to the test substance.  Therefore, these statement have
been removed from the report.	i, 1-2, 4-2, 4-3, and 4-5

Peer Reviewer – Schlesinger

The study was conducted in the proper manner and the results presented
support the conclusions reached. There were no protocol deviations that
would have affected the study outcome.	No action required

	Some specific comments are as follows:

p. 3-6. It is stated that the exposure period was "...at least 6 hours
per day." How much longer than 6 hours did exposures last?	Statement
revised to six hours per day	3-6

p. 3-8. The last paragraph in the Statistical Analysis section notes
that both raw and transformed percentages were used in statistical
tests, but that transformations were not reported since they were not
statistically significant. I presume that the raw percentage data were
also not statistically significant. The correct procedure would be to
perform any needed transformations, perform statistical testing on the
transformed data and then only reports results of these tests.
Transformed data added to the report.	3-9, F-2

p. 3-9. Why were some tests performed at one level of significance and
others at two levels? Appendix I. Table 1-6. There is no indication of
the actual median size (MMAD) of the aerosol noted in the chamber nor of
its concentration.	Added explanation of statistical significance in
statistics section.

MMAD not calculated due to the relatively small concentration of aerosol
(also suspected to be animal dander and fur)	3-10

Peer Reviewer – Goldsworthy

The only potential treatment related statistical difference between
control and treated groups was an increase in unossified sternebrae in
the high dose group on a fetal basis. This finding was not statistically
increase on a litter basis, the basic unit of analysis. Additionally,
increased abnormality was noted in the last forming sternebrae that
exhibits the most variable appearance in normal specimens. Collectively,
it is appropriate to not consider this finding in the NOAEL setting for
developmental toxicity. However, text should (1) provide a table with
the unossified sternebrae data in the results section for both fetus and
litter basis (Section 4, pages 4-1 to 4-3), (2) clearly state on 4-3,
skeletal observations, that the statistically significant increase was
noted on a fetus basis, and (3) consider noting this finding in the
abstract (as it was noted in the Summary and Discussion sections).	All
references to the unossified sternebrae removed from the text.  The
revised statistical analyses did not identify this as a statistically
significant observation.	i, 1-2, 4-2, 4-3, and 4-5



Based on the maternal toxicity findings in the 20,000 mg/m3 group, I
concur that the NOAEL for maternal toxicity in this study is 10,000 m
m3. This NOAEL setting considers the slight transient decreases in feed
consumption at the 10,000 mg/m3 exposure level not to be considered
adverse. I concur with the NOAEL developmental toxicity to be 20,000
mg/m3 ; this level of exposure did cause an increase in the incidence of
unossified sternebrae on a fetus basis but was not statistically
significant on a litter basis.	No action required

	Text/Editorial Comments

Discussion, page 4-5: Top paragraph, last sentence - for clarification,
insert “in feed consumption” following “that those decreases”.
Statement revised	4-5

Appendix F - Uterine Implantation Data (page F-2): Wrong number of
significant figures used for mean and standard deviation data.	Corrected
F-2

Provide treatment group information when referring to Dam IGL353 on
pages 4-2 and 4-3.	Exposure group added	4-2, and 4-3

Consider adding summary tables in result section on (a) fetal
implantatiodweight data and (b) the most frequently noted skeletal
observations. Along with the Table addition for unossified sternebrae
data, such Tables would aid the reader in assessing the overall data and
providing support for the developmental toxicity setting.	No change

	211 (b) Research Group Reviewer 

General Comments

- When the report is final, it should have sequential numbering in
addition to the A-1, A-2, etc. system so that any reader can be certain
that all pages are present.	Sequential pagination added	All pages

- The QA page for this report, as for the previous ones, needs to state
what was audited. This needs to be distributed and reviewed by the
sponsor before the report can be finalized. Providing a blank QA page
with the audited report is not acceptable.	QA statement added	ix



- There are two issues with regard to statistical analyses. First,
transformations were performed and therefore need to be reported,
whether or not the results are statistically significant. Second, the
report ignores statistically significant linear regression wherever the
multi-group comparison is not statistically significant. If the lab is
to proceed with this approach, it needs to spell out the criteria for
ignoring significant linear regressions. It also needs to address these
findings in the text of the results.	Results of transformed data
reported.  Results of linear trend test added to the results	F-2, 4-1

- Appendix J provides information about the composition of the vapor to
which the animals are exposed. This information is pertinent and should
be discussed in the text. In the current draft, there is no mention of
these results.	Added to the exposure data results.	4-4

- The Historical Control Data, presented as Appendix K, is a great
improvement over the previous version. Several recommendations:	No
action required

	- The lab might want to rethink inclusion of maternal body weight and
food consumption intervals in this data; I've never seen it presented in
any other lab's HC data, and haven't found a need for it previously for
data interpretation. Keeping it or not can be the lab's call. I strongly
urge them, however, to put the body weight data into order, and to
footnote the first page, K-1 , to explain that the data collected at the
Annandale facility is presented separately from the East Millstone
facility.	Maternal body weight and food consumption removed from the
historical control data.  All non-Annandale data removed from the
historical control data	Appendix L

- I also recommend that the lab lighten the background on lines within
the data, since it makes the information difficult to read.	Background
lightened	Appendix L



- The usual use of a historical control database, other than to
demonstrate the lab's recent experience, is to aid in interpretation of
questionable findings. To make it easier for the data to serve this
purpose, it should be organized by external, visceral, and skeletal, as
the lab has done, and then by parts: i.e., adrenals, discolored, and
adrenals, hypoplastic, should be in order together rather than in a
group of diverse discolored and/or diverse hypoplastic findings. This is
in part because certain observations can be related due to having a
similar embryologic cause: there's not much difference between a
dumbbell shaped and a bifid centra, one may simply be more ossified than
the other.	Data reorganized	Appendix L

- The one area of statistically elevated fetal findings in the study,
unossified sternabrae, should be compared to the historical control data
range as part of the discussion of interpretation.	This incidence of
this observation in the treated groups was not statistically
significantly different from the control incidence in the revised
statistical analyses.

	Specific Comments (by page and section)

Page i:

Third sentence: ng/m3 should be mg/m3	Corrected	i

Page ii:

Table of Contents: Page 2-1, there are two headers on the page between
Study Initiation and inlife Test Period. Either all	Corrected	ii

should be listed, or Experimental Start Date and Experimental
Termination Date should be single-line entries within another section.
Added Experimental Start Date and Experimental Termination Date to Table
of Contents

	Page viii:

Quality Assurance Statement: This should have been virtually complete
when included in the draft report. The information of what was audited
and when will need to be provided to the sponsor prior to the
finalization of the report.	QA statement added	ix

Page 1 -1:

Summary, first paragraph: The lab hasn't indicated why 2000 mg/m3 was
expected to be a NOAEL.	Statement replaced with the Sponsor-supplied
statement	1-1



Page 1-2:

Summary: "The only fetal observation that may be related ..." The report
has thrown this statement out as though having a single finding is
insufficient for establishing a NOAEL, but the finding might have been
caused by treatment. If the laboratory does not believe that the
increase in unossified sternabrae is related to exposure, then, by
inference, this should be considered a random event that can be
discounted because: not statistically elevated when the litter is used
as the statistical unit; close to the upper range of the historical
control data; no other indications of delayed ossification, etc. The
second paragraph indicated that this was evidence of developmental
toxicity. The report needs to be clear on whether this is interpreted as
developmental toxicity or not. If it is, then the developmental NOAEL
should be called at 10000 mg/m3.	Summary revised to reflect the revised
statistical analyses where none of the observations are statistically
significant	1-2

Page 2-1:

Justification for selection of test system: I believe that the rat was
specified in the vehicle emissions testing document (1994). It is not a
required species in the developmental testing guideline, it is a
recommended species.	Revised	2-1

Page 2-2:

Justification of dose selection: The high dose& one-half of the LEL.
Statement changed to the statement provided by the Sponsor	2-2

Page 3-1:

Test Substance, Supplier: The correct name may be Chevron Products,
Richmond Refinery OR Chevron Research and Technology Company, but not
Chevron Research. Please review the shipping records; if these state
Chevron Research, there will need to be a correction.	Name changed	3-1

Characterization of the test substance: At a minimum, this section
should mention the analytical report in Appendix J.	Added reference to
Appendix J	3-1

Page 3-4:

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

Page 3-8:

Statistical Methods: Transformations, since they were performed, ought
to be reported, statistically significant or not.	Results of statistical
analyses on transformed data added to Appendix F	Appendix F

Page 3-9:

Statistical Methods, continued: There should be explanations provided
for: (a) why linear regression is performed when there isn't a
difference between groups; (b) what criteria was used to ignore
statistically significant results from linear trend tests in these
circumstances. What was the criteria for performing a lack of fit test?
Linear regression tests run automatically in our statistical package. 
The lack of fit test also runs automatically with our statistics package
to determine if the linear regression test was appropriate for the data.
 The statistically significant linear regression results have been added
in the results.	4-1

Page 4-1:

Clinical inlife observations and survival, second paragraph: Clinical
signs included alopecia of the trunk in the control and 20K groups, and
alopecia extremities in the 1 OK group. Please correct the sentence.
Gestation body weight: GD 5-8 and 0-21 C BWC were both statistically
significant for linear trend. This should be mentioned and discussed
briefly in the text, unless an adequate explanation is provided in the
methods section (p. 3-9). Gestation food consumption: GD 0-21 and 5-8
were both statistically significant for linear trend. Same comment as
for gestation body weight.	Revised	4-1

Page 4-2:

Fetal observations: Appendix F (F-2) also contains total malformations,
total variations, and total affected, and so needs to be referenced here
along with Appendix H.	No change.  The Appendix F data is reported under
the uterine implantation data section.  The total malformations, total
variations, and total affected would not be discussed in the fetal
observations section

	

Page 4-3:

Visceral observations: Fetus No. IGL353-2, the conjoined twin, should be
described before the findings in other fetuses (clarifies the single
incidences of malpositioned carotid and subclavian arteries, which this
fetus had also). "Misshapen spleen" should either be classified as a
variant or a malformation. Skeletal observations, second sentence: The
most frequently noted observations were rudimentary lumbar ribs (79),
bifid thoracic vertebral centra (24), and dumbbell shaped thoracic
centra anlage (28), not advanced sternabrae (20). The sentence would be
correct if revised to "The most frequently noted skeletal ossification
variants were ...." "Advanced" should be clarified for both the
sternabrae and the calcaneus (such as Calcaneus, advanced ossification)
Visceral section revised.  Misshapen spleen classified as a variation.  

Skeletal section revised

	4-3

4-3

Page 4-4:

Exposure Data and Chamber Conditions: The analytical report in Appendix
J should be referenced and discussed here. Table 4-1 : Mean analytical
concentration for the 10,000 mg/m3 group was 10,327 mg/m3 (p. 1-8, 1-1
7), not 10,319.	Analytical report referenced and discussed.  Mean
concentration revised for 10000 mg/m3 group 	4-4

Final paragraph: The minimum noise level was 72.2 db (512112003, p.
1-21), not 72.5 db.	Corrected

	

Page 4-5:

Discussion, first paragraph: The report needs to be consistent in the
interpretation of maternal results. In this first paragraph of the
discussion, the report has reiterated that there were statistically
significant decreases in body weight gain, but goes on to indicate that
these decreases were not biologically significant. If they are not
biologically significant, then the maternal NOAEL should be 20,000
mg/m3, not 10,000 mg/m3. The study director needs to make a decision as
to whether the effect upon body weight gain is an indication of adverse
effect or not, and present this position consistently and clearly.
Second paragraph: The report provides a justification for why the
increased incidence of unossified sternabrae is not deemed to be
evidence of developmental toxicity. The report should further back up
this position with regard to the data within the historical control
base. The first sentence conveys the perception that this observation
may be related to treatment. If that is what the director believes, this
is an appropriate way to word this sentence. If, as suggested by the
subsequent dismissal of this finding, the study director doesn't believe
this finding to be due to exposure, the sentence should objectively
(rather than subjectively) state that the only statistically significant
fetal observation was an increased incidence of unossified sternabrae.
Discussion revised to clarify that the decreased body weight change was
an adverse event and to reflect the revised statistical analyses of the
skeletal findings.	4-5

Page 4-6:

Protocol Exceptions, chamber temperature and humidity: The values for
the deviations should be listed herein. All of them.	Section revised. 
All deviations are presented in Appendix I	4-6, I-23 – I-30

Page 5-2:

Fetal external and visceral examinations: Does the laboratory have
criteria for what observations constitute malformations and which are
variations? Criteria needs to be provided for why an observation
wouldn't belong in one of these two categories.	Criteria added	5-2

"Misshapen spleen" should be considered one or the other.	Classified as
a variation

	Appendix I, page 1-3:

Third paragraph, second sentence: "...injecting a weighed amount of the
test substance.. ."	Corrected

	Page I-11:

The standard deviation for Group 2 chamber temperature is 1.6 (p. 1-15),
not 1.7.	Corrected	I-10

Appendix K, pages K-2 to K-5:

The studies are misordered.	Pages reordered and move to Appendix L

	211 (b) Research Group QA/QC Reviewer

The following items in the report and associated raw data require
further consideration:

Page vi, 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.	Sponsor added to the compliance page	vii

Page vi, 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, it should be included in the sponsor’s compliance
statement.	We do not consider this a GLP deviation.  We have added this
to the Test Substance section.	3-1

Page viii, Quality Assurance Statement:

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

Page 1-2, Summary:

 The No Observable Adverse Effect Level for developmental toxicity was
indicated to the 20,000 mg/m3 even though there was statistically
significantly reduced unossified sternebrae at that level. It appears
that the laboratory’s historical control for this variation is well
below this level as well. Please comment.	No change.  The revised
statistical analyses did not indicate that the incidence of unossified
sternebrae in the treated groups was statistically significantly
different from the control group.

	Page 2-1, Experimental Termination Date:

 There were re-evaluations much later than the date listed here. There
were re-evaluations (indicating data were recorded) as late as 10/8/02.
Please clarify.	Date changed to July 19, 2002 based on the data from the
skeletal examinations.  The skeletal data and the analytical data were
the only two components of the study where data could be collected
directly from the study (from EPA definition for the experimental
termination date) after the in life phase was completed.  All other
entries were calculations using the collected data or corrections to the
data.	2-1

Page 3-3, Age at Initiation of Gestation:

 It appears that animals were approximately 14 to 15 weeks of age at
this time. Please clarify.	Revised to 14 to 15 weeks	3-3

Page 3-1, Test Substance:

 The original test material receipt record was not with the rest of the
study data during the audit. Please verify that there is a record
indicating the 9/10/01 receipt date.	The date of receipt was recorded in
the test substance usage log.

	Page 3-1, Test Substance:

 An expiration date of September 30, 2006 (5 years after receipt) is
given for the test substance. Are there stability data to support this
expiration date?	No change.  Expiration date applied as per EMBSI SOPs.

	Page 3-2, Particle Size Analysis, first sentence:

 The determination was made once during the chamber trials from the
control and 20,000 mg/m3 concentrations.	Sentence revised	3-2

Page 3-5, Environmental Conditions:

 The ranges given for animal room temperature and humidity are
protocol-specified ranges. The actual measured ranges should be
reported, giving relative times and extents parameters were out of
range.	Actual ranges added	3-5, 4-6



Page 3-7, The Test Atmosphere:

 The last sentence should indicate “generally” within 10% of target
exposure………..	Revised	3-8

3Page 3-8, Examination of Fetuses:

 The handling of fetus #2 from the low exposure group dam #353 should be
explained (i.e., it had both skeletal and visceral exams done).	Added
3-9

Page 4-2, External Observations:

 It would be helpful to indicate that this animals was a 2000 mg/m3
animal.	Added	4-2

Page 4-3, Visceral Observations:

 No exposure relationship (or lack of) is indicated for these findings.
Clarified in the first paragraph of the fetal observations.	4-2

Page 4-4, Table 4-1:

 For the 10,000 mg/m3 group, the analytical column of numbers should
read, 10,327, 280.2, and 9762 (last number is correct as is), I believe.
Please verify.	Corrected	4-4

Page 4-5, Discussion, second paragraph:

 Historical control data should also be discussed in relation to the
unossified sternebrae.	Discussion of unossified sternebrae removed to
reflect the results of the new statistical analyses.	4-5

Page 4-6, Protocol Exceptions:

 It should be indicated that the three temperature excursions were in
the control chamber. The high humidity levels in the chambers really did
not relate to the low temperatures. The high humidities were noted on
the majority of days in the control chamber and only sporadically in the
other groups.	Protocol exception revised	4-6

Appendix D, GD 5-20 and GD 0-21, Group 1 values:

 When comparing these values to the values printed out in the
statistical analyses, they differ. Recalculating by hand confirms that
values used for statistical evaluations are correct. These are GD 5-20,
361.5, +37.4; and for GD 0-21, 505.6+49.6. Please clarify.	The correct
values are 361.5 ± 36.6 for GD 5-20 and 505.6 ± 49.6 for GD 0-21.  
D-1



Appendix F, % PreImplantation Loss and % PostImplantation Loss, Groups 1
and 4:

 The values for Group 1 PreImplantation loss in the statistical analyses
were 7.9+17.7. Recalculation by hand confirms this. As well, the values
for the Group 1 and 4 Post Implantation loss is 4.4+10.2and 2.2+3.2. The
value for the Group 4 PreImplantation loss should be 3.5+3.9. Please
verify and correct if necessary.	Group 1 Preimplantation loss corrected.
 All other values were correct as reported.	F-2

Appendix H, Fetal Observations, Incidence of Fetal Observations, Page
H-1:

 The Total Litters with Skeletal Variations entry for the 0 mg/m3 should
be 17/25, correct? There were 25 evaluated and the statistical print-out
indicates 25. The Value for the Total litters with Skeletal
Malformations should be 1/25, I believe. Please verify.	Statistics
rerun.  There were only 24 litters with skeletal evaluations (one litter
only had one fetus, which was used for visceral evaluations)	H-1

Appendix H, Fetal Observations, Incidence of Fetal Observations, Page
H-1:

 The Total Litters with External Malformations in the 20,000 mg/m3 group
is indicated in the statistical print out as 0/25. This should be 24,
correct? Please verify.	Corrected	H-1

Appendix H, Fetal Observations, Individual Fetal Observations, Page
H-17:

 The “a” finding is indicated in the data, but is qualified by
“other” in the key. It appears that additional information is
needed. This finding is not included in the “Incidence of Fetal
Observations”. Please clarify.	No change.  Page H-17 appears fine

	

Appendix H, Fetal Observations, Individual Fetal Observations, Animal
numbers 394, 402, 411, 382, 353, 433, 399, 452, 375, 369, 410, and 424
all are listed as “Sternebrae VI – “Advanced” when they should
be “Misshapen”. These have two different codes used on the data
sheets. Please verify and correct as necessary. Summary tables also need
to be corrected.	Corrected	H-5, H-17 –H-19, H-40, H-46, H-51, H-73,
H-74, H-86, H-91, H-98, and H-104

Appendix H, Fetal Observations, Individual Fetal Observations, Animal
number 353:

 I could not find the skeletal observations designated as footnotes W,
DD, m, and R in the raw data. Please verify these findings.	M CORRECTED
IN FETUS 2	H-51

Appendix H, Fetal Observations, Individual Fetal Observations, Animal
number 442:

 Is there a finding missing from the report for this animal? Please
compare raw data to that reported for this animal.	Corrections made in
the data

	Appendix I, Table I-3, 1st Summary Table, Group 2, S.D. of the Chamber
Temp.:

  Should be 1.6.	Corrected	I-10

Appendix I, Table I-3, 1st Summary Table, Group 4, Minimum of the Rh of
the chamber:

 Should be 49.	Corrected	I-10

Appendix I, Table I-3, Group 2, May 3rd, first hourly exposure:

 Should there be an * by this value as it appeared that an alternate IR
was used to check the low value. The miran used was #5901. Was this
miran calibrated? The one used for other checks (number 5879) was
calibrated, but I could not find documentation that 5901 had been
calibrated. Please clarify.	No * needed.  The data is from the GC.  The
MIRAN was used to eliminate the GC as the problem, which it did.  I
don’t know why an uncalibrated MIRAN was used, but I would expect the
values of the two MIRANS to be within±10% of each other which was
sufficient accuracy in this instance..

	Appendix I, Table I-3, Group 3, April 29th, second hourly value:

 Should be 11,120. Please verify.	Corrected	I-15



Appendix I, Table I-3, 1st Summary Table, Group 2, May 13th, mean
exposure in mg/m3 should be 1943.	Corrected	I-10

Appendix J, Results, page J-2:

 It would be helpful to present the neat MRD-00- 716 values for
comparison to those of this study.	No change.  The characterization data
is presented in a separate report.

	Data Findings

Test Material Transfer and Usage Records:

 The data indicate that the small tanks used in this study were labeled
1A, 2A, 3A and 4A. The SOP for small tank filling indicates that the
small tanks will be assigned the large tank number (i.e., 2), followed
by a sequential letter each time they are filled or re-filled (i.e., 2a,
2b, 2c, etc.). Since tank labeling for this study did not follow the
SOP, this should be addressed as an SOP deviation in the study records.
Memo added to data

	Animal number 475, a group 2 female was recorded at sacrifice with 4
implants and 10 corpora lutea; Animal number 469, a group 3 female was
observed with 2 implants and 6 corpora lutea. These should have been
verified, per SOP, but were not. This should be handled as an SOP
deviation.	Notes added to the data and the SOP was revised to clarify
the intent of the SOP

	Animal #370, fetus #13 was observed with numerous external
malformations. Cleft palate was noted and “confirmed” at skeletal
exam, and then the finding was crossed out. Did the technician mean to
write “not” confirmed at skeletal exam? Please clarify.	Note added
to data

	

Animal Room Log for Room 103:

 There were study animals from study 171634 in this room from April to
May and June 2002. As of June 17, study number 171734 occupied this
room. Then study number 171834 animals were in the room. It is not clear
when one study was over and the other begins. It appears that there are
still animals from the first study in the room. The Animal Husbandry
Record goes through 6/12 for study number 171634 changing to study
number 171734 on June 14th. Please clarify as all 171634 animals should
have been sacrificed the end of May (around the 23rd).	This information
is in the Room Check Book for PE 103 on the Extreme or Unusual Animal
Observations form.

The study numbers were removed from the Husbandry Record.  This was
optional information that was confusing due to the males from the Study
171634 shipment of animals that were used for Study 171734.

	The memo found in the 211 Clean Air Act Program Study File concerning
the 4/12/02 change in GC column needs to go to the 171634 data book. The
entire data trail concerning the GC malfunction and subsequent
resolution needs to be followed to make sure everything is documented
properly. The memo previously mentioned, indicated that the GC column
was replaced 4/12/02, yet there is another memo that indicates the GC
began to malfunction on 4/12/02 and was serviced on 4/18/02 by EMBSI
Engineering and Electronics personnel (valves changed), but that
didn’t solve the problem, so the column was replaced on 4/19 and
recalibrated on 4/22. The GC calibration data sheet indicates that the
actual vs. nominal ratio was not good, so the GC was recalibrated on
4/22. The nom/act. ratio wasn’t really that far off from what one
expects, so the reasoning behind these actions is unclear.  Presently,
it is not clear in the data that there was a malfunction and how it was
detected. Please clarify.	Memo added to the calibration and trials book.
 

As the data states the difference between the actual and nominal was
poor.  In the high concentration the difference was greater than 10%
which was considered unacceptable.  There had been intermittent problems
with the actuator and valves and  they were presumed to be the problem. 
The rebuild of the actuator and valves did not resolve the problem, so
the  new column was installed.  This reduced the difference between the
actual and nominal to less than 10%.

	

Particle Size Data:

 The data sheet for the 20,000 mg/m3 group indicates the calculation for
the concentration of particles = 2 mg/m3. I believe 20 is correct. That
is what is reported.	Corrected

	Charcoal tube samples were stored frozen prior to transfer to the
analytical laboratory for desorption and analysis. Are stability data
available to support that the samples are stable under these conditions
for this period of time?	No change.  These are standard methods for
handling these types of samples.  Additionally, the data from these
samples shows that the samples were stable.

	Analytical Data, General Comment:

 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 printout 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.).	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.

	GC Printouts, General Comment:

 Throughout the GC data, the individual performing the analysis is
identified only as “Inhalation Staff”. Since more than one
technician can perform the exposures, the responsible technician must be
identified in the data. It was noted that there is a dated signature on
the chromatogram cover sheet that is identified as a review signature.
Is the reviewer also the operator in all cases? Please address/clarify
in data.	Memo added to the Section 211 notebook

	GC Printouts, Butane Standard:

 Calibration Injections are labeled with “Room Air.” These need to
indicate “butane standard” and acceptable ranges for these butane
standards need to be given.	Memo and procedure added to the Section 211
notebook

	

211(b) Toxicology Research Group

Gas+ETBE Rat Developmental Toxicity Study Report 

Reviewer Checklist

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