    Final Report Whole-Body Inhalation Developmental Toxicity Study in Rats
         With Gasoline with MTBE Vapor Condensate (MRD-00-713: 171334)
                                       
           211(b) Research Group Response to EPA Second Draft Report
                         Comments dated June 24, 2008
                                       
EPA June 24, 2008 Comment #1:
Although the inferential statistics now used to analyze the fetal examination data are valid, the report tables still inappropriately show fetal incidences.  As currently presented, these data are still difficult to properly evaluate.  Table 4-2 and Appendix H should present the group mean + SD values for the percentage affected per litter.
RG response to EPA June 24, 2008 comment:
The RG considers this to be a valuable comment, and acknowledges that it would be desirable for the tables to reflect the litter data.  However, the appendices contain the full data set, including the results of the statistical analysis by litters.  Further, it would be a lengthy procedure for the laboratory to revise report tables as EPA has suggested, as such revisions would require programming changes.  Programming changes require computer validation and subsequently also require additional Quality Assurance review.  These tasks would need to be completed before the report could be resubmitted for QA review as required under GLP.  For those reasons, the work group has not requested that the lab revise the report as described in this comment.  However, the results can be presented in the suggested format at later stages of data utilization, such as publication/risk assessment, where the change would not delay completion of the 211(b) testing requirement.
EPA June 24, 2008 Comment #2:
The results/discussion section should address the dose-related response for rudimentary lumbar ribs.
RG response to EPA June 24, 2008 comment:
The following text was added to the Results.
There was a dose-related increase (not statistically significant) in the number of fetuses with rudimentary ribs.  However, this was not considered toxicologically significant because the fetal incidence in the 20,000 mg/m[3] target group (6.04%) was just slightly higher that the historical control range (5.52%) and the litter incidence (20.8%) was less than the historical control range (25.0%).  Additionally, the litter incidence did not show a dose-related increase.
EPA June 24, 2008 Comment #3:
Although some fetal-examination endpoints are combined for analysis as shown in Appendix K, there are additional sets of observations that warrant a combined analysis.  In particular, dumbbell-shaped and bifid anlage of thoracic centra are clearly related findings and neither occur in the control group.  Their combined incidence should be evaluated statistically.  On a similar note, cartilage-based observations should be combined with related ossification findings.  For example, page K-3 lists a combined analysis for hypoplastic vertebral anlage centra (item 31) and a combined analysis for hypoplastic vertebral centra (item 32).  These two sets of findings, one for cartilaginous anlage and one for bone, are clearly related; their combined incidence should be evaluated statistically.
RG response to EPA June 24, 2008 comment:
We agree with the comment that additional combined statistical analyses could be appropriately performed.  Dumbbell-shaped and bifid anlage of centra are combined in Skeletal analysis #31.  The combined statistical analysis for skeletal analysis 31 and 32 would be unlikely to alter results, as there was only one litter affected in each of the low and middle exposure groups, and the litter at the low exposure group was already included in the analysis for hypoplastic vertebral anlage centra.

EPA June 24, 2008 Comment #4:
Some findings listed in Appendix H still do not clearly denote an abnormality.  In particular, "presacral vertebrae" does not indicate an abnormality.  Appendix K, in combining the findings of cervical and lumbar presacral vertebrae, indicates that these denote "extra presacral vertebrae." This should be clarified in Appendix H as well.
RG response to EPA June 24, 2008 comment:
The individual table lists the "presacral vertebrae" as "extra presacral vertebrae" in the individual data (the location is also given).  The summary data before the individual data lists the findings as "extra cervical vertebrae" or "extra lumbar vertebrae".
The remaining noted observations were also revised.

EPA June 24, 2008 Comment #5:
The authors should consider classifying the finding of an extra cervical vertebrae as a malformation rather than a variation.  Although variations in the number of thoracic and lumbar vertebrae are not uncommon, changes in the number of cervical vertebrae are rare.  Note that this does not affect study interpretation as this finding was observed only in a low dose fetus that, based on other findings, was already regarded as malformed.
RG response to EPA June 24, 2008 comment:
For GLP compliance, the laboratory must record observations according to the classification designated in their SOP.  However, we agree in principle with the comment.
In the report, there is a note with the individual data for this fetus that discusses the laboratory SOP.  
