Document ID: EPA-HQ-OAR-2003-0065-0576
Agency: epa
Document Type: Supporting & Related Material
Title: 
Posted Date: 2010-05-06T04:00Z

Inhalation Toxicity Report
                                   Comments
                                Action/Response
                                    Page #
"Baseline Gasoline plus Ethyl Tertiary Butyl Ether Vapor Condensate: A 13-week Whole-body Inhalation Toxicity Study in Rats with Neurotoxicity Assessments and 4-week In Vivo Genotoxicity and Immunotoxicity Assessments (by Huntingdon Life Sciences, Study No. 00-6129m Sponsor No. 211-ETBE-S).
EPA 

ORD finds that in general, the main study and the specific neurotoxicity, genotoxicity, and immunotoxicity assessments included in the report were conducted according to the EPA test guidelines.  At this time, however, we cannot recommend approval of the study report as is, because we have identified significant deficiencies in the statistical analyses and reporting of data in the functional observational battery (FOB) and motor activity assessments.  Similar issues were described previously in our memorandum to you of June 20, 2002, regarding the subchronic toxicity study for baseline gasoline.  In addition, we are recommending that more data be collected for the micronucleus assay.  The attached memorandum provides specific comments on the study report. 

This was the original draft report of these BG+EtBE VC studies and did not include the revised statistics for sister chromatid exchange and neurobehavioral endpoints recommended by EPA after reviewing the Baseline Gasoline Vapor Condensate submission.  The revised statistical evaluations have been completed and are included in the second draft report of this study.

Additional data were collected for the micronucleus study and findings are described with the study review. 

Our review was conducted by the ORD 211(b) Health Effects Team, comprised of scientists from our National Health and Environmental Effects Research Laboratory (NHEERL) with assistance from the National Center for Environmental Assessment (NCEA) and the Office of Science Policy (OSP). 

ORD reserves judgment on the interpretation of the results, and conclusions drawn from them, until a formal assessment of the 211(b) findings and any other pertinent information is completed at a future date.  We strongly encourage the RG to address our comments before final versions of the reports are submitted to EPA.  The RG should also adequately respond to the comments raised by the independent peer reviewers and provide the additional information requested.

No comment needed

At your request, NHEERL scientists have reviewed the submissions from the American Petroleum Institute (API) entitled, "Gasoline ETBE Vapor Condensate: A 13-week Whole-body Inhalation Toxicity Study in Rats with Neurotoxicity Assessments and 4-Week In Vivo Genotoxicity and Immunotoxicity Assessments."  The report was submitted in three volumes from Huntingdon Life Sciences (Study No. 00-6129, Sponsor No. 211-ETBE-S) with additional reports from other subcontractors to API, including reports on neurotoxicity assessment using GFAP, immunotoxicity, and genotoxicity assessment using sister chromatid exchange and micronumcleus assays.  In addition, NHEERL reviewed the reports of two peer reviewers of the studies and the API response to the peer review comments.

While in general the NHEERL reviewers concluded that the studies were conducted in accordance with the testing guidelines, there were also significant exceptions involving the conduct of some of the studies and analysis of the data.  These issues were described previously in our memorandum to you of May 21, 2002, regarding the baseline gasoline study.  We understand that these continuing issues will be addressed by API in a revised final report.  In addition to the issues previously noted, we have the following assessment of the gasoline plus ETBE study reports.

Background information.  No comments needed

Sister Chromatid Exchange Assay and Micronucleus Assay
The micronucleus assay appears to be properly conducted according to the required guidelines.  The conclusion in the report that "G+ETBE VC may cause an increased incidence in micronucleated polychromatic erythrocytes" is supported by the results.  However, given the fact that the significant outcome was somewhat dependent on the statistical analysis method used, it is appropriate to collect data from a second set of slides.  This is being done, and it is hoped that the addition of the data acquired will make the interpretation firmer.

A second set of slides from the micronucleus assay was read and the entire report revised to reflect the additional data.  Although there was an apparent increase in group mean micronucleated immature erythrocytes (mie) with increasing concentrations in the second set of slides, none of the values from animals exposed to BG+ETBE Vapor condensate were statistically significant compared to negative controls.  The report has been re-written as draft 2 to include these data.

The sister chromatid exchange (SCE) assay appears to be conducted according to the required guidelines and the conclusion that BG+ETBE VC did not induce SCE is supported by the data.  There is one proviso  -  as with other studies in the BG set  -  namely, that additional statistical analysis (1-tailed Dunnett test) be performed, since there was some concern with reliance on a trend test given the nature of the data.  These additional statistics spear to be in progress.

Additional statistical analysis was performed and included in draft 2 of the report.

Immunotoxicity
We concur with the review by Drs. Schlesinger, Goldsworthy, Twerdok, as well as with the results obtained by Dr. K. White, who performed the immunotoxicity evaluations.  The data indicate that the exposure to Gas+ETBE vapor at 10,000 and 20,000 mg/m[3] suppresses the antibody-forming cell response, compared to vehicle control.  This suppression of the antibody response to sheep erythrocytes occurred in the absence of any decrease in body, spleen or thymus weight.  As such, the data indicate that the immune system in rats, exposed at the two highest doses, is unrelated to a generalized overt toxicity and suggests that GAS-ETBE may target the immune system.

A minor point is that Dr. Goldsworthy, in his review of the "Immunotoxicology Evaluation...," stated that "Gasoline+ETBE vapor condensate did result in a dose-related decrease in the spleen IgM antibody-forming cell response to sheep erythrocytes."  In fact, there was no "dose-related" decrease, since the IgM AFC/10[6] and the IgM AFC/spleen data demonstrated that there was a slightly more pronounced suppression in the AFC at 10,000 mg/m[3] versus that at 20,000 mg/m[3].  This is observed in the data presented in Fig. 4 and Table 2.

These findings were reported to EPA under Section 8(e) in 2002.

The EPA reviewer is correct.  No changes were made in the report.

FOB and Motor Activity
This protocol has been reviewed previously with the studies for baseline gasoline, and gasoline containing MTBE, ethanol and Tame, and the same issues apply here.  After the first baseline gasoline study, we discussed those issues with the sponsor and the contractor, and they are being addressed.  However, since these studies were actually conducted prior to those discussions, the changes have not been made in the current document.  Therefore, the outstanding issues are only summarized here.

Revised statistics for neurobehavioral endpoints were performed and Draft 2 of this report rewritten accordingly.

Overall, the study was conducted in compliance with the published EPA test guidelines, with the exception that some endpoints (e.g., reactivity measures, gait score, sensorimotor responses) are not ranked, as is required.  The statistical analysis of the neurobehavioral data is not acceptable in its present form, but the sponsor's statistician is working on more appropriate analyses.  Specifically, both motor activity and FOB data should include repeated measures, and grip strength and foot splay duplicates should be averaged and the analyzed.  Sex should be included as a factor in the overall ANOVAs for all endpoints. 

See comment above

A review of the actual data revealed no obvious differences across treatment groups.  There were a few differences pretest, but these were small in magnitude and were not evident with subsequent testing.  Provided that appropriate statistical analyses do not raise new questions, we accept the results of the study.

Application of revised statistics did not alter the conclusions of these tests.  No neurobehavioral effects were observed.

Neuropathology
We have examined the neuropathology sections of the study, and based on the information provided, we conclude (a) the neuropathologic assessment was conducted in accordance with the EPA Neurotoxicity Screening Battery Guidelines and (b) the data are consistent with the conclusion of the report that there is no evidence of treatment-related neuropathology in the 20,000mg/m[3] exposure group.  The reviews by Drs. Goldsworthy and Schlesinger are consistent with our evaluation.

No comment needed

General Conclusions
In general, it appears that the studies were conducted in accordance with the appropriate testing guidelines.  The study reports were prepared before API received EPA comments on the reports from the baseline gasoline vapor condensate 13-week study, and so a number of issues raised in our review of that study recur in the current report.  We understand that those issues will be addressed in a revised report, in action to the issues raised here specific to the gasoline plus ETBE 13-week study.  Overall, we agree with the study, the peer reviewer comments and API's response to those comments.  Among the issues that still need to be addressed for the gas plus ETBE study are the analysis of second set of slides in the micronucleus assay and additional statistical analysis for the SCE assay, both of which appear to be planned or are currently being done.

See comments above.

Peer Reviewer  -  Schlesinger

Overall, the main 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.  However, a number of concerns related to statistical analysis and the basis for interpretation of toxicological significance are indicated.

The conclusions are generally sound, although in one specific case there could be some misinter-pretation of results.  This involves the conclusion in the main study section of the report that the 10,000 mg/m[3] level is considered to be the NOAEL.  However, there seem to be some effects on some parameters at this exposure level from results reported in the main study, and there clearly is an effect at this level in the plaque forming immunological assay.  An individual reading just the main summary may, thus, arrive at a false conclusion as to the biologically effective level of the vapor.

As noted previously, it would have been preferable if all of the satellite studies employed, to the extent possible, comparable statistical approaches to data analysis.  They basically all had the same study designs and could have used similar statistical assays.  This would have avoided the potential for different statistical techniques to have different sensitivities.

The conclusions reached in the micronucleus study are generally supported by the data presented.

The conclusions reached in the GFAP study are supported by the data although the authors seem to imply that the results are quite nondefinitive.

The conclusions reached in the SCE study are supported by the data.

The conclusions reached in the immunological assay are supported by the data.

Detailed comments for each of the draft reports are attached.

The statistically significant clinical chemistry changes at 2000mg/m[3] cited by Dr. Schlesinger tended to occur in one sex only, a treatment related pattern was not apparent and absolute differences were small with no corroborating histopathology.  The Immunotoxicity study was performed with animals exposed for 2 - weeks during the 13-week study and data were analyzed separately, demonstrating significant effects at 10,000 and 20,000mg/m[3].  The "main study" conclusions reflect only the results and NOAEL of the subchronic toxicity study.  A table has been added to Draft 2 summarizing the results of all satellite studies so that the reader will be fully apprised of NOAELs for different endpoints.  All satellite studies are supplied as Appendices to this report.

While recognizing the value of this comment, the RG must employ different contract laboratories with recognized special expertise to perform satellite studies with different endpoints.  Each laboratory employs an individual statistical package developed by experience and the historical database for the assay.  Since most of these packages are computer-based, it would be resource intensive and costly to require the subcontractors to employ statistical methods that are not standard practice in their laboratory.  In addition, comparison of current data to historical databases within a laboratory, where appropriate, would be more difficult if statistics were not consistent.

MAIN STUDY

Section 2.20.1  Method of Analysis (p. 43)
When the Bartlett's test indicated nonhomo-geneity 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 would have made the statistical analysis of all data sets much more internally consistent and would not present any potential for different types of tests having different degrees of conservativeness in detecting any significant changes from air control.  In any case, the investigators should indicate which specific statistical tests were used for which datasets.

A single computerized statistical testing package was employed for all parameters listed on p.43-44 of report.  In this package, analyses by individual sex or sexes combined were programmed to be performed separately by one-way ANOVA, rather than analyzed together by two-way ANOVA.  Terminal and recovery data were also analyzed separately.  After discussion with the study director, it was decided that additional justification for the statistical method employed routinely for repeat-dose studies throughout this testing facility was probably not needed.

The use of the one-way ANOVA as described infers that when there was more than one time point for analysis, e.g., terminal and recovery, that data for each endpoint was analyzed separately at each of these times.  It would have been possible to use one evaluation that could have included the various time points and both genders in each analysis for each endpoint.  A clearer justification for the statistical methods actually used by the investigators should be provided.

See comment above.

What was the rationale for performing statistical tests at two levels of significance.  In the satellite studies, for the most part only one level was used.  This difference could lead to interpretational problems.

Contract laboratories provide both levels of significance to satisfy needs of a range of clients and both values are often identified in standardized statistical packages for computers.

Section 3.1.  Chamber Monitoring.  (p. 47):
The table on page 48 shows that the air control chambers as well as the test chambers contained particles.  What were these and why were there not appropriate filters on the chamber intake to remove particles from the incoming air?

The mean particle distribution was comparable between controls and treatment groups.  Appropriate filters were in place but some particles are invariably present in chamber atmospheres, representing background air.  

While the data support the conclusion that the gasoline atmospheres were in vapor form and not in particulate form, has any thought been given to the possibility that the small "background" particles which were also present in the vapor atmospheres could have acted as modalities for the condensation of the of the vapor and a vector to areas it may not have reached if inhaled in the absence of such particles?

It is unlikely that test vapor would condense on these particles, given the volatility of these light end vapor components, and the high level of consistency in constituents proportions demonstrated analytically from week to week.  In addition, no unique particle disposition was observed microscopically in lungs or nasal passages.

Furthermore, from Appendix A it appears that the particles have a wide size range, from <1 to >9 micrometers.  Finally, it is stated on page 48 that the results "   indicated that the atmospheres were essentially vapor only...since there was no substantial difference between the test substance chambers and the air control chambers for particle content.  This statement does not seem to make sense.  What the data show is that all chambers had some particles.

It is agreed that the data showed that all chambers had some particles.  The sentence in question should convey that the absence of higher particulate mass in weekly air samples from test substance chambers compared to control chambers supported the likelihood that introduction of BG+ETBE vapor condensate did not contribute additional particulate material into the chamber atmospheres, and thus could be considered all vapor.

On page 47, mention is made of the possibility of some inaccuracy in the calibration of the IR monitor.  This would be an unacceptable source of experimental error, since exposure atmosphere monitors should be accurately calibrated prior to the start of any study.

Average nominal concentration varied slightly above analytic concentration (low and mid doses; 2,000mg/m[3] and 10,000mg/m[3]), to slightly below analytic concentration (high dose; 20,000mg/m[3]).  Nominal concentrations are calculated by measuring the weight differences in the 5 gal. cylinder from which the test sample is dispensed, divided by the total vol. of air (m[3]) passing through the chamber.  Large differences between nominal and analytical concentrations are useful in identifying possible leakage in sample delivery or chamber distribution, and could suggest disposition of test material on chamber walls, reducing the amount of material in the breathing atmosphere.  Where variations are small, significance is questionable.  In this study, differences are small and the analytical concentrations very closely match the target concentrations, providing a reliable determination of exposure.  Calculation of nominal concentration can be most affected by the accuracy of calibration of the airflow rate, making calculation of nominal concentrations a less accurate measure of chamber concentrations than analytical determinations.   The calibration of the IR monitor is the more accurate calibration than the chamber airflow calibration because the IR monitor calibration involves the precise injection (using gastight syringes) of fixed volumes of test substance into a known and fixed volume of air.  By comparison, the chamber airflow calibration involves the measurement of large volumes of air (~200 LPM) using a mass flow meter, a procedure that is inherently less accurate.
Summary and R/D clarified that nominal and IR results were in `acceptable' agreement.

Section 3.8.1. Hematology (p. 50)
It is stated that exposure related differences seen at week 4 are no longer apparent at the terminal sacrifice interval.  However, examination of the data suggests that there still is some decrease in reticulocyte count in females at the terminal interval sacrifice.

The decrease in reticulocyte counts seen in high dose females at terminal sacrifice was not statistically different from controls probably due to the large standard deviation and thus was not considered to be significant.  According to the study director, the absolute differences in reticulocyte counts for high dose females compared to controls decreased from 18% at wk 4 to 9% at wk 13, resulting in a decrease in statistical significance.

Section 3.8.3. Clinical Chemistry (p. 51)
A statement in this section seems to imply that if statistically significant changes occurred in only one gender then they would be considered to be toxicologically insignificant.  This precludes the potential for gender-specific real effects.  Also, some of the significant differences in these parameters seem to result from wide variability in the standard deviations of values rather than from any differences in mean values.

The statement on p. 51 of the report concerning discounting the effects seen in only one sex was not meant to diminish the potential significance of gender-specific effects in general but only to indicate that, in instances of random clinical chemistry changes, isolated occurrences in only one sex was one parameter for rejecting statistical significance alone as biologically important, especially if a treatment related pattern was not apparent, absolute differences were small with wide standard deviations and there was no corroborating pathology.  

Section 3.9 Organ Weights (p. 51)
Organ weight data presented in Table 12 appear to have been analyzed by two different statistical procedures, and some values are significant with one of these and others are significant with the other method.  Firstly, why were two tests used and secondly, how is the reader to interpret significance obtained using two different methods on the same dataset?

According to the study director, organ weight data are analyzed by parametric procedures using Dunnett's test when data are homogeneous and by the Modified t-test when data are non-homogeneous.  Data for each organ parameter [absolute wt, relative wt compared to body wt or brain wt] is evaluated for homogeneity independent of other organs and the appropriate statistical test is applied to comparisons of all dose groups within that parameter.  In this study, statistical significance was determined by a non-parametric test only once, in the data set for male lung wt relative to body wt at terminal sacrifice; the relative weight at 20,000mg/m[3] was significantly higher than controls.  The absolute lung wt in high dose males was not increased significantly.  Dr Schlesinger had suggested that transformation of data when variances are not homogeneous and subsequent use of parametric tests throughout could make analysis of all data sets more consistent [see response for Section 2.20.1].  The RG believes that, in lieu of using transformed data, the laboratory' approach for evaluating homogeneous and non-homogeneous data sets by separate appropriate methods within each parameter is a reasonable method for determining statistically significant changes in each organ.  

Peer Reviewer  -  Goldsworthy
 

The potential inhalation toxicity of G-ETBE was assessed via whole body exposures to male and female rats for 13-weeks followed by a 4 week recovery period.  In this report, standard toxicology neurotoxicity parameters were presented (genotoxicity and immunotoxicity results were presented in separate reports; reviews presented below).  The design of the study was appropriate and sound and the study was properly conducted.  The report was clear and comprehensive.  The deviations noted had no effect on the study results or data integrity.

No comments needed

Exposure techniques and procedures were adequate to test the potential toxicity of gasoline-ETBE.  Exposure levels of test substance were relatively close to target levels although differences between measured and nominal concentrations were greater than the expected 1:1 ratio.  Although the causes of the differences were not determined, it was hypothesized that differences may have been due to problems with chamber airflow and the IR monitor.  Animals were exposed to all major components of gasoline-ETBE in their expected proper portions.  Week to week data consistently was noted as well, indicating stability of test substance and proper techniques.

Experimental data revealed no effect of G-ETBE on survival, feed consumption, clinical observa-tions, or gross abnormalities at necropsy.  No exposure-related effect was observed on functional observation battery or motor activity; no effect was observed with ophthalmoscopic assessments.  There were no exposure-induced differences in absolute body weights.  The 20,000 treated female rats exhibited decreased body weight changes between exposure weeks 4-12.  There were no observed exposure-related effects in coagulation values at 4 weeks and terminal intervals.  High dosed animals exhibited decreased reticulocyte counts at 4 weeks; this was not seen at the terminal sacrifice.  Similarly, various high dose exposure-related differences in clinical chemistry values at 4 weeks were not present at the terminal intervals.  High dosed exposure-induced increases in the kidney weights of male rats were considered to be the only exposure-related toxicological effects.  Renal microscopic findings in male rats were consistent with hydrocarbon-induced nephropathy; this species and gender specific alteration is generally considered no relevant for human risk assessment.  I concur with the study conclusions and the NOAEL setting of 10,000 mg/m[3] exposure level. 

See response to Dr. Schlesinger comment on Section 3.1 Chamber monitoring  -  IR monitoring.

211(b) Research Group Reviewer

EPA has requested that the proportion of oxygenates in each gasoline/oxygenate blend be added to reports [see BG+EtOH comments, p. 2 under Immunotoxicity, 6/9/03].  To be accurate and consistent across studies, the % oxygenate in the starting liquid and % oxygenate in vapor condensate or at least % oxygenate in vapor condensate could be added to the text [2.4 Test substance] of each subchronic report.  This data is available in the Analytical Appendix but reviewers do not always consult that section.

The RG has decided that this information will be provided in a separate report addressing composition and analyses of all test samples.
[Paula, Lorraine  -  is this the current thinking?]

13 Week Subchronic Study:
Dr. Schlesinger noted that since the Immunotoxicity assay showed positive results for BG/ETBE at 10000 and 20000mg/m[3], a NOAEL of 2000mg/m[3] should be considered for the subchronic study of which the Immunotixicity study was a part. [A similar issue arose for the positive results in the GFAP assay of BG/EtOH.]  Assignment of overall LOAELs and NOAELs will be determined by the 211(b) workgroup.

A table has been added to Draft 2 summarizing the results of all satellite studies so that the reader will be fully apprised of NOAELs for different endpoints.  All satellite studies are supplied as Appendices to this report.

Summary:
p.6,  1[st] paragraph, next to last sentence:  major components were assayed once per chamber per week.  Please add "per week".

Added.

p.6,  2nd paragraph, final sentence:  Please add that control rats as well as 20000mg/m3 rats were examined histopathologically at terminal sacrifice, and that Recovery were not evaluated histopathologically. 

Added.

p.6,  QA requested that variation between nominal and analytical concentrations be specified.  Variations were 4-12%; both values were close to or slightly above target concentrations.

Not added since the difference was minimal and to be consistent with prior reports in this test program.

p.7,  The change in creatinine kinase is a decrease, not increase.

Revised to decrease.

Methods
p.19, 2.5.6  Weight at Initiation of Exposure:  Please verify that weights for female animals in the Immunotoxicology satellite group are the same here as in the Immunotoxicology report Appendix B.  According to QA there is some mix-up with these data in that report [item 17, Audit dated 1/13/03]

Body weigths reviewed and report appears to be correct as is.

p.29, 2.12.5  Feed Consumption:  Is it correct to assume that feed consumption was calculated for each animal and then averaged for the dose group?  See item 21 in QA report [Audit 1/13/03].

The reviewer is correct.

p. 36, 2.16  Immunotoxicity
Line 7: please spell sensitized with a z, rather than s. 
Done.

p. 43, 2.20 Statistical Analysis: Report states that Mean Clinical pathology values were evaluated statistically only on termination data.  Since when?  Statistics in all 211(b) studies were run at both wk4 and at termination.  Tables indicate this as well.  Pleas check and correct [cited by QA also, Audit 1/13/04].

Revised.

Descriptions of additional neurobehavioral analyses, recommended by EPA, will be added to this section.

Done.

Results and Discussion
Restate somewhere in the Results that animals were treated for a minimum of 65 exposures over 13 weeks, perhaps at the end of the chamber monitoring paragraph [p. 47].  Peer reviewers have requested confirmation of the number of exposures.

This has been added to the Summary on page 6 and the M/M on page 15.

p.47, 3.1 Chamber Monitoring:  Since QA requested that the variation between nominal and analytical concentrations be specified in the Summary, the information belongs here as well.  The variations were 4-12%, both values were close to or slightly above target concentrations.

Not added since the difference was minimal and to be consistent with prior reports in this test program.

p.50, 3.7 Neurobehavioral Studies:  Any changes in interpretation due to application of revised statistics requested by EPA will be addressed.

Added to report.
p.
p.50, 3.8.1 Hematology and Coagulation (Table 9):  Please add the following entries here and, if appropriate, in the Summary:

-  At week 4, statistically significant (p<0.05) increases were seen in MCV for males at 10000 and 20000mg/m[3]; mid dose was slightly higher than high dose value and effect was not seen at termination.

Added to report.

-  Decreased reticulocyte counts similar to those seen at week 4 were also present in females exposed to 10000 and 20000mg/m[3] at termination but were not statistically significant because control values were also lower [cited by Peer reviewer, see API submission to EPA, 7/15/04].

All values were lower at termination than at interim interval  -  this is normal change in maturing rats.  Therefore, no discussion added to the report.

p.50, last sentence:  Add "no significant exposure related differences..........."

Added to report.

p. 51, 3.8.3 Clinical Chemistry (Table 11):  line 7 [2nd sentence].  Please correct.  Change in creatinine kinase over three doses is a decrease, not an increase that is statistically significant at 20000mg/m3.

Revised report.

Chloride values were statistically significantly increased (p<0.01) at wk 4 in females but the increase was the same over 3 doses and was not observed at termination.  Although the data indicates a plateau effect and was not seen at wk 13, a significance level of p<0.01 should be mentioned and probably discounted as not biologically significant.

Added to report.

211(b) Research Group QA/QC Reviewer

A raw data and draft final report review was performed to assure compliance with GLP regulations (Part 79.60).  One hundred percent of the text and summary tables of the report were verified and at least 10 percent of individual data were verified.  The following were reviewed and inspected:

1.	Protocol

2.	Draft Final Report dated June 19, 2002

3.	Red Spiral Binder labeled A-12, 00-6129, Gasoline ETBE Vapor Condensate, A-12, 00-6129, Gasoline ETBE Vapor Condensate: a 13-Week Whole Body Inhalation Toxicity Study in Rats With Neurotoxicity Assessments and 4-Week In Vivo Genotoxicity and Immunotoxicity Assessments, Volume II, Book 17 of 18 Section: Charcoal Tube Monitoring Record.

4.	Clear Binder labeled Study Title:  Gasoline ETBE Vapour Condensate, A-12, 00-6129, Gasoline Tame Vapor Condensate A 13 Week Whole-Body Inhalation Study in Rats with Neurotixicity Assessment and 4-Week In Vivo Genotocixity and Immunotoxicity Assessment, Study Director:  Gary M. Hoffman, B.A., D.A.B.T; Scientist: Samuel R.Onanuga, M.S., Associate Director; Tox-Support Chemistry: Kay Saladdin, B., Book: 1 of 2
Sections: Exposures, Method Development, Invalid -- Raw Data

5.	Clear Binder labeled Study Title:  Gasoline ETBE Vapour Condensate, A-12, 00-6129, Gasoline Tame Vapor Condensate A 13 Whole-Body Inhalation Study in Rats with Neurotixicity Assessment and 4-Week In Vivo Genotocixity and Immunotoxicity Assessment, Study Director:  Gary M. Hoffman, B.A., DABT, Scientists: Samuel R. Onanuga, M.S., Associate Director, Tox-Support Chemistry: Kay Saladdin, B.S. Book 1 of 2
Sections: Abbreviations, Information, Protocol, Analytical Procedure, Pipette Calibration, Method validation, Pretest Trials, Exposures

6.	Clear Binder labeled A-12, 00-6129 Gasoline ETBE Vapor Condensate:  A 13-Week Whole-Body Inhalation Toxicity Study in Rats With Neurotoxicity Assessments and 4-Week In Vivo Genotoxicity and Immunotoxicity Assessments Volume II Box 3 of 8
Section:  Charcoal Tubes

7.	Clear Binder labeled A-12, 00-6129, Gasoline ETBE Vapor Condensate: A 13-Week Whole-Body Inhalation Toxicity Study in Rats With Neurotoxicity Assessments and 4-Week In Vivo Genotoxicity and Immunotoxicity Assessments Volume II Book 2 of 18
Sections 1 Body wts, food wts and Clinical Observations

8.	Clear Binder labeled A-12, 00-6129, Gasoline ETBE Vapor Condensate: A 13-Week Whole-Body Inhalation Toxicity Study in Rats With Neurotoxicity Assessments and 4-Week In Vivo Genotoxicity and Immunotoxicity Assessments Volume II Book 2 of 18
Sections:  Computer Raw Data Changes and Additions Edits, Daily Environment and Viability Records, Clinical Observation Study List, Computer Sort

9.	Clear Binder labeled 00-6129, Clinical Pathology Stats ::Hematology:: Coagulation :: Clinical Chemistry
FOB stats:: Foot Landing Splay ::Grip Strength :: 

10.	Clear Binder labeled  Clinical Pathology Raw Data, Table of Contents, Sponsor Code: A-12, Study 00-6129, Book 1 of 1, Volume:
Sections:
    I.	Signature Page, Raw Data 	correction codes, Protocol parameter page, Memos

    II.	Transfer sheets
    III.	Clinical Chemistry
    IV. Coagulation

11.	Clear Binder labeled A-12, 00-6129, Necropsy Data Book 1 of 1

12.	Chamber Monitoring Records

13.	Test Material Usage and Dispensing Logs

14.	Test Material Receipt Records

15.	GFAP Raw Data

16.	Eye Exam Observations

17.	Motor Activity Data

18.	FOB Observations

19.	Animal receipt Records

Background  -  No comments needed

The following items are comments or require further consideration:

1.	Page 2, 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.  It should also include those non-GLP compliant issues that the Testing Facility has indicated. 

Added to report.

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

Added to report.

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

Added to report.

4.	Page 4, Quality Assurance Statement: Since HLS has been inspected all of the subcontractors (except CDC) these facility inspections should be listed on 

The inspections were generic and not study specific and therefore will not be listed on the QA Statement.

5.	The date that the QA report of the report draft and study data was reported to the Study Director and management needs to be added to the QA Statement.

Added to report.

6.	Page 6, Summary:  When indicating the different reports (various places in the report), they should be noted as "appended" to this report.  They are stand-alone reports, but since they were requirements of the protocol, they need to be reported with the rest of the study.

Added to report.

7.	Page 6, Summary, first paragraph, last sentence: Major components were assayed once per chamber, per week.

Added to report.

8.	Page 6, Summary, second paragraph, next to last sentence: Histopathological evaluations of all selected or prescribed tissues were conducted on all control and 20,000 mg/m[3] exposed.......Lungs and gross lesions were also examined for mid dose animals at the Terminal interval.  Recovery animals received..........

Added to report.

9.	Page 6, Summary, third paragraph: are the values reported for chamber concentrations grand means and standard deviations for both chambers?  They appear to be the mean of the means and standard deviations of the two chambers.  Please clarify.

10.	Page 6, Summary, third paragraph, third sentence: Some idea of the extent of the variation between nominal and measured concentrations should be given.

The values reported for chamber concentrations are grand means and standard deviations for both chambers as explained on page 48.

Not added since the difference was minimal and to be consistent with prior reports in this test program.

11.	Page 7, Summary, third paragraph, reporting of body weight findings:  Due to the manner in which the body weight changes were calculated (always the change from 0 to all intervals) it is difficult to tell if there were body weight changes at one interval or all of them.  If they had been reported by interval, 0-1, 1-2, 2-3, etc. one may see that there were no differences throughout the 12 weeks.

Not practical due to computer system limitations.

12.	Page 7, Summary, paragraph 4, second sentence: it appears there was a decrease in creatine kinase values instead of an increase.  Please clarify.

Revised report.

13. Page 8, Summary second paragraph, first sentence: The results section indicates this sentence indicates that no gross abnormalities clearly related to test substance.... Please clarify.

Revised report to clarify `exposure-dependent incidence of bilateral renal foci of red discoloration'.

14.	Page 17, Date Received: There is a record of cylinders being received on 9/10/01.  Were these empty cylinders?  Please clarify.

Cylinders were only received on 17Sept01 and this included 4 large cylinders full of test substance and 20 small empty cylinders.

15.	Page 17, Expiration Date:  It should be indicated that the test substance stability was tested concurrently with the study and the results showed it to be stable for the duration of the study.  (See Appendix S?)

Added to report.

16.	Page 18, Supplier:  Are Kingston and Stoneridge, NY the same place.  Animal records indicated Stoneridge, NY.

These are equivalent Charles River locations.

17.	Page 19, Weight at Initiation of Exposures (Grams):  The values for Immunotoxicity will have to be corrected when these values are corrected in the report tables.

BW's reviewed and appear to be correct as is.

18.	Page 20, Veterinary Care:  It seems that any conditions requiring veterinary care need to be reported whether they are felt to be non-test substance-related or not.

No change required, information is maintained in raw data if needed.

19.	Page 23, Environmental Conditions:  Some indication of the extent of the excursion should be given, ie, only occurred on 11 days in one of the high-dose chambers, etc.

Temperature excursions were minimal (+2C) and need not be detailed further.

20.	Page 28, Ophthalmoscopic examination:  Shouldn't it be noted that animals with ocular findings were not used for study.

This was noted in Section 2.6.
Page 19
21.	Page 29, calculation: is the denominator the body weight (kg) of the group or the individual?

The denominator is the body weight (kg) of the individual and then the values are averaged.

22.	Page 32, Relative Humidity:  Please verify the actual range, I found 40-64% to be the range.

Data checked and range is correct as reported.

23.	Page 36, Micronucleus Evaluations:  It is indicated that the positive control animals were sacrificed with CO2, but nothing for the test and control-treated inhalation/exsanguinations.

These are the same animals as for the SCE evaluation as explained on prior page and their disposition was explained there.

24.	Page 42, GFAP Evaluations:  It is indicated that animals were sacrificed with CO2 inhalation, but I believe they were anesthetized with CO2 and sacrificed by decapitation.  Please comment.

OK as is  -  they were killed by CO2 and then decapitated immediately for tissue collection.

25.	In the report test, Page 43, Section 2.20 Statistical Analysis states that mean clinical pathology values "only run on term", but mean values were run at 4 weeks and term.

Report corrected.

26.	Page 51, Clinical Chemistry, second sentence:  It appears there was a decrease in creatine kinase values, not increase.  Please verify,

Report corrected.

27.	All pages in the tables and appendices do not contain the Sponsor No. 211-ETBE-S.  The hematology, coagulation and clinical chemistry individual appendices and summary tables should be page numbered 

Not done for previous studies in this series.

Full report will be paginated for final report.

28.	Table 8, Summary of Functional Observational Battery:  Group 3, Forelimb Grip Strength, Trial 2, Pretest Interval.  Please verify the mean and standard deviation.  I get 845.5+-149.8.
Table 9 has been revised to include mean of the 2 trials (grip strength and foot splay).  All averages have been recalculated accordingly.

29.	In Table 2, Page 6 and 7, data summaries appear to be from the satellite groups for this study.  Individual data was not included in Appendix B to support these summary data.

Pages were misplaced and have been moved.

30.	Table 7, Motor Activity Values, Summary of Statistical Analyses: Second sentence should indicate that there was a recovery period only for certain animals.

This entire Table 7 has been replaced by the new statistical report from the EMBSI statistician Mark Nicolich.  New report included in 2nd draft.

31.	In Table 11, Week 4, Group 3M 10,000 mg/m[3], the statistics were performed with an n of 9; the mean was 0.07889 with a standard deviation of 0.0232.  Table 11 reports an n of 10, mean 0.07 with an SD of 0.0233.  The statistics should be rerun using all 10 values.  The values in Table 11 appear to be correct.

The statistics were re-run with all 10 animals in group 3 males.

32.	Different Ophthalmologists examined the animals pretest and at 13 weeks.  This should be indicated in the ophthalmology report (.e, which data was observed by each Ophthalmologist.  It does appear in the listing of personnel.

This is shown in Table 3 by the summary letters from the 2 different examiners.

33.	Table 14, Lesion Incidence Summary with Expanded Severity Levels:  The preface to this table does not include an explanation of NAD.  Please verify that NAD is defined somewhere in the report.

Added to preface page.

34.	Table 14, Lesion Incidence Summary with Expanded Severity Levels:  For tissues that were examined and found normal, why is there no indication of normalcy on the summary table?  Should NAD be entered where there are no findings?

NAD is defined on the Preface.  Only if there are findings in some animals does NAD print for the rest.  No change can be made to the manner in which the program operates.  No change to report. 

35.	Table 14, Appendix M:  There is a comment of Atelactasis for several animals.  Is this something that should be included in the summary tables, even though it is probably an artifact?  It is not included.

Comments are not included on summary tables and are limited to the individual data pages.  No change to report.

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

Data has been added to Appendix Q.

37.	Appendix A, Chamber Monitoring Results:  For the Particle Size Determinations, Total Mass Concentration, the standard deviation is printed as 0.0.  Actual values need to be included.

Number of decimal places in the mean GSD has been revised to match mean values presented in the same table.

38.	Appendix A, Chamber IIIB, the nominal value for Nov 7 should be 10.200, I believe.  Please verify.

CMR revised (all affected satellite reports revised as well).

39.	Appendix A. Chamber IIA, second value for Dec. 11.  Please verify that the value is 2240 and not 1510 mg/m3.
 
The value of 2240 is correct (this represents an average of the 1[st] and 2[nd] duplicate sample and was calculated according to SOP).

40.	In Appendix B, Page 10, Animal 5062 -- The edit change on Day 27 for this animal and Animal 4061 should be reviewed.  The finding "red nasal discharge" that was observed for Animal 4062 (seen previous week and following week) was edited out and two findings that were recorded for Animal 4061 were added to Animal 4062.  Should all three findings be present for Animal 4062?

Raw data corrections reviewed and it appears to be intentional that red nasal was removed as an incorrect entry.  No change to report.

41.	In Appendix D, individual data is reported from Week-1 to Week 17.  Appendix E reports weight change from Week 0 to Week 17.  Should the weight change from Week-1 to Start of Study (Week 0) also be reported in Appendix E?

No - body weight change is calculated and presented from baseline (Week 0).  No change to report.

42	The Preface, Page 5 of Appendix H for abnormal movements does not match the key in the data notebooks.  A clarification to the raw data key is necessary to explain convulsions, tremors, or fasciculation as abnormal movements in order to match the key as being reported in Appendix H.

Key in data lists "Abnormal Motor Movements"; convulsions, tremors, or fasciculation are listed on data pages under "Motor Movement" heading; data appears to be clear as is.

43.	Appendix H, Individual Functional Obs, Battery Ev.:   Several animals were noted in the data with tremors when handled.  This is not reported, (animal numbers, 4572, 3568, 1578, 3565).

Table has been up-dated.

44.	In Appendix J, Group 3, Females, PT value  -- The individual values for Animals 3553 and 3554 records "EM" meaning equipment malfunction, results not valid.  The raw data records no plasma.  An explanation is needed to clarify the "no plasma" recording versus "equipment malfunction."

Data indicates QNS due to EM, therefore reporting the value as EM appears to be accurate.  No change to report.

45.	In the clinical chemistry values, Appendix K, Week 4, Animal 3054 -- The reported values for TBIL  -  0.03 and DBIL 0.04.  This results in n IBIL value of -0.01, but the table reports 0.0 as the value.  Should this value be footnoted and explained?

The reported value of 0.0 is OK and will be footnoted with explanation that a negative value is not possible.  Footnote "Result was -0.01 but presented as 0.00 since a negative biological value not considered realistic" added to report.

46.	Appendix L needs to be page numbered.

Report will be paginated for finalization.

47.	Appendix S, Analytical Report:  The table on Page 5 of the analytical report does not record that samples were collected on 12-14-01.

Added to the report.

48.	Appendix S, Analytical Report:  The required signatories as listed on Page 2 of the analytical report should sign the analytical report.

This will be done when the final report is prepared.

49.	Appendix S, Analytical Report:  The table on Page 7 of the analytical report records ND for the total from columns Samples Group 1A and Samples Group 1B.  All other tables in this report record 0.00 for the total of these samples.  The 0.00 should be used for consistency.;

Corrected

50.	Appendix S, Analytical Report:  The four figures in the analytical report should document that the chromatograms were obtained from samples collected during Exposure 3.

Corrected

51.	Appendix S, Analytical Report:  In Figures II, III and IV of the analytical report, 15 peaks were labeled.  The peaks for 2,3 dimethylpentane, 2, 4 dimethylpentane, 2 methylpentane and trans-2-pentane are not labeled.

Printout is accurate but not all peaks are labelled on the printouts due to printing constraints where peaks, such as those noted in the comment, are compressed into each other.

52.	Appendix S, Analytical Report:  In Figure III of the analytical report, the response time for the peaks 2,3-dimethybutane and benzene do not agree with the values as reported on the raw data chromatograms.

Printout is accurate but not all peaks are clearly labelled on the printouts due to printing constraints where peaks, such as those noted in the comment, are compressed into each other.

53.	Appendix S, Analytical Report:  In Figure IV of the analytical report, the response time for the peaks 2,3-dimethylbutine, n-hexane, and benzene do not agree with the values as reported on the raw data chromatograms.

Printout is accurate but not all peaks are clearly labelled on the printouts due to printing constraints where peaks, such as those noted in the comment, are compressed into each other.

54.	Sister Chromatid Exchange Assay Report, Page 9:  The last line contains a redundancy.  I believe the 68 hours can be deleted.

OK as is- this is correct per protocol.

55.	Sister Chromatid Exchange Assay Report, Page 9:  Can it be verified that the sodium heparin tubes stability had not expired?

I do not have an answer to this query from Bioreliance but assume that they would not have used expired sampling tubes.

56.	Sister Chromatid Exchange Assay, Page 9:  The lot number for Cyclophosphamide, 108H0568 is the same from the two previous studies, but they all have had different expiration dates.  Could the same lot have different expiration dates?

Yes  -  the expiry date was extended by the manufacturer.

57.	Sister Chromatid Exchange Assay Report, Page 19:  The mean SCEs per cell for animal number 5052 should be 22.2 not 21.7, I believe.  Please verify.

Report corrected.

58.	Sister Chromatid Exchange Assay Report, Page 20:  The AGT per group value for Group 1 appears to be 26.6 not 26.  Please verify.

Report corrected.

59.	Immunological Evaluation Assay Report:  The body weights, body weights gains, food consumptions and resulting mean tables have animals mixed up.  All groups are represented under any group number.  This means also that the means for groups is not correct.  These animals need to be rearranged and data needs to be in the correct table.

Issue corrected by HLS IT and pages have been  re-run.

60.	It also raises the concern that this should not have happened.  The computer should not let animals of different groups be summarized together.  Please comment.

Per HLS IT, 1 file was not copied to a particular environment with the rest of the data; issue has been rectified.

61.	Immunological Evaluation Assay Report, Page 9, Table ES-1:  It is assumed that the column titled "Maximum Effect" is there to indicate that the largest effect was seen in the 10,000 and 10,000 mg/m3 levels the reader can gather this.

Yes  -  the maximum effects were seen at the middle exposure level.

62.	Immunological Evaluation Assay Report, Page 11, second sentence starting with Cyclophosphamide:  This sentence is a repeat of the first one beginning with Cyclophosphamide.

Corrected

63.	Immunological Evaluation Assay Report, Page 13, Terminal Body Weights and Organ Weights:  It only gives a weight for the control animals.  Shouldn't it compare the controls to the dosed groups?  (Note: This mean may change when body weight tables are corrected).

Sentence listing only control weights deleted.  The treated groups were statistically compared to the vehicle control group as per protocol and this report.

64.	Immunological Evaluation Assay Report, Page 16, second sentence:  The decrease in the IgM-antibody-forming cell response was not dose-related when evaluated as total spleen activity, so the term dose-related should be deleted.

The term has been deleted.

65.	Immunological Evaluation Assay Report, Page 17, second sentence:  This sentence should probably indicate the "immune" functional ability of the animals.......

The sentence has been corrected and it now indicates "humoral immune" functional ability of the animals.

66.	Immunological Evaluation Assay Report, Page 17, last sentence:  Shouldn't it be indicated that there was an adversely affected humoral immune response of female SD rats "at levels of 10,000 and 20,000 mg/m3?"  And that there was a no effect level?  This should also be indicated on page 8, the Executive Summary.

This has been corrected in both locations.

67.	Immunological Evaluation Assay Report, Page 2, GLP compliance statement:  The sentence indicating that the "method used for antibody-forming cell assay had not been formally validated" needs to be clarified.  It was my understanding that the assay was validated and that producing a formal written procedure for doing so was ongoing.  Please indicate this in the GLP compliance statement.

Corrected.

68.	GFAP Report:  It is indicated that the "spirit of GLPs" were followed.  It would be useful to indicate where the study fell short of full GLP compliance.
Chris Sexsmith to prepare a listing of deficiencies.

69.	GFAP Report, QA Statement:  It is indicated that the findings of QA reports were reported to the Study Director and Management.  Was this Gary Hoffman, the Study Director and His Management at HLS?  That is the way it should be as there can only be one Study Director in a Study.

Gary Hoffman is Study Director.

70.	GFAP Report, Page 8, second sentence:  There is not a Two-Generation study to be done with ETBE.  It will only be a One-Generation study.  As well the next sentence should indicate that this study was to use the GFAP assay for assessing the effects in a subchronic study.  The in-life animal data should also be referenced.

ETBE changed to MTBE.

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

Changed first sentence to: "Typical GFAP assay values obtained at this laboratory for different regions of the rat brain are presented in Fig. 2 (i.e. historical data)." And a citation for these results is now mentioned in the Figure Legend (p. 21)  and reference section (Martin and O'Callaghan, 1995).

72.	GFAP Report: For Tables 5 and 6, the group 4 female and male pituitary values are based only on one animal so should probably not be included in the mean table.

Pituitary data removed from these summary tables.

73.	GFAP Report, Tables 5 and 6:  Values for Cortex in Group 4 males and females appear to be switched or incorrect.  Male value should be 1.72+-0.07 and the female value should be 1.76+-0.06.  As well, the cortex value for the Group 1 females SEM should be 0.08, and the SEM for the Hippocampus value for the Group 3 males should be 0.22.  Please verify.

Corrected mean values. SEM values are computer generated so okay as is.

74.	GFAP Report, Table 7, The Pituitary values reported for the group 4 males and females are not correct.

Values are corrected.

75.	GFAP Study:  It appears that there was a large coefficient of variation in the data and that is the reason why many sample analyses were repeated.  There needs to be a detailed explanation of this in the data, and repeat values need to be footnoted as such.  Actual values reported need to be clearly indicated in the data.

Usual laboratory experimental procedures; Repeated GFAP on sample if %CV is > 15% then include the values that are <15% CV in the report. No comment necessary.

76.	Micronucleus Report, Page 15, second paragraph:  It appears that the group mean value of 2.4 is within the group mean historical control range.  Please verify.

The highest value included for group mean values in the vehicle historical control data is 2.1, therefore the value of 2.4 is outside the vehicle historical control range and the report is correct.

77.	Micronucleus Report, Page 16:  Should the conclusion indicate that the possible increase in frequency of micronuclei was only in females?

No, we don't think the conclusion should be changed, as values for all females were within the individual historical vehicle control range and the increases observed in the group mean incidences were not dose related and not sex specific.

78.	Pathology Report:  For all animals were only gross observations were reviewed microscopically, the pathology table indicates, "Tissues w/o comment under gross observations were within normal limits," and "The following tissues were unremarkable microscopically, no tissues examined", yet the tissues with gross lesions were examined.  It is confusing.  Please clarify.

This refers to all other tissues aside from the ones with gross lesions.  No clarification needed.

DATA Findings:

1.    In the GFAP data book, it is indicated that Christine Mason is the API Principal Investigator.  This should be corrected.

Data clarified.

2.	There appear to be numerous tissues missing and damaged from the Group 1 animals.  Is there an explanation for this?  Many recuts were requested and sometimes tissues were not found.  Please comment.

We have reviewed the raw data and do not find the number of recuts or damaged tissues to be excessive compared to other studies from this prior timeframe or our current timeframe.

3.	Appendix S, Analytical Report Data:  The titles on the raw data notebooks for the analytical analysis of the charcoal tubes should be corrected:  the titles states TAME and the study is ETBE, several words in the name of the study were misspelled, and the second notebook should be labeled Book 2 of 2, not 1 of 2 as the first notebook was labeled.

Corrected.

4.	Analytical Report Data:  Freezer stability for the charcoal tubes is necessary in the method validation to support the data from the charcoal tubes being frozen after collection and before extraction (one day and 13 days storage).

Freezer stability was not conducted and will be noted on the GLP Compliance Statement.

5.    Analytical Report Data:  The charcoal tubes collected on 12-14-01 were analyzed and the data was rejected.  New samples were collected on 12-17-01 and the results from this analysis were reported.  Documentation is needed in the data to explain why the data from 12-14-01 was rejected and the location of any data that was obtained during the analyses of the 12-14-01 samples.  This rejected data should be included in the notebook with the explanation of the situation.  The data states "instrument problems and response" but the documentation does not record what the instrument problems were and what was done to correct the situation.

No samples were injected due to an instrument error. The details of the instrument error were not documented.

6.	Analytical Report Data:  In the analytical raw data for Exposure 35, the sequence of samples records incorrect sampling numbers.  The sample number states 12 samples, but 14 samples were analyzed.  Sample 5 is listed incorrectly three times in the sequence.

Correct as is. The sequence was manually corrected.

7.	In the clinical chemistry data a note should be made to clarify that for the GGT parameter  -  when the results is a  - 1L alert, -2L alert, or -3L alert, the value is reported as 0 and the sample is not rerun.  All other instrument flags result in the sample being rerun.  This procedure should be clarified.

A memo has been added that explains the SOP requirements for reporting values below the assay linear limits.

8.	In a discussion with the Supervisor of Clinical Pathology, the SOP CP1.2.22 details the requirements for performing and reporting a manual differential versus the automated reading for the differential.  The supervisor stated that the technician would review the raw data and if the % Monocytes is greater than 7, or the % Eosinophils is greater than 5, or the % Leucocytes is greater than 5, a manual differential count would be performed.  If the manual differential count confirms the automated values, the automated values would be reported.  If the manual values were different than the automated values, the manual values would be reported.

Correct as is. 
SOP procedures indicated that this is correct.

The problem of performing statistical calculations using the absolute values obtained from the manual differential with the automated values was also discussed.  The supervisor stated she would consider removing the absolute values from a manual differential from the statistics since the number of cells observed are different.  But the % values should be included in the statistics since the number of cells observed is used in the calculation of the percentages.

The SOP in place at the time of analysis required that the manual differential count and the calculated absolute counts be reported. The LUC (U%) are counted by the Advia but manual differential does not count LUCs, therefore LUCs were reported as 0.0.

9.	The one-generation protocol for Baseline Gasoline and TAME is enclosed in the pharmacy file for this study.

Protocol has since been removed.

10.	Test Material Inventory Records:  Large Container number 1 should have 235,348 g not 224,063.  Please verify.

Data has been corrected.

11.	Test Material Inventory Records:  There are records indicating two #1 containers that are empty.  As well, a sheet for study 6129/4203 indicates sample #1 returned to sponsor on 3/15/02, but container #1 was returned to Chevron on 12/7/01.  Please clarify.

We  have reviewed the records and they clearly indicate that only 1 container #1 of G/ETBE was returned on 7Dec01.  The 15March02 shipment was for G/ETOH not for G/ETBE.

12.	Lactose Dehydrogenase values, particularly for the control animals, appear very irregular.  Are there any explanations for this?  

It is known that LDH levels in rat are highly variable. A review of the data did not indicate any unusual findings with the assay itself. QC results were within range. Sample processing record did not indicate any anomalies either. The handling of the animals during bleeding may have contributed to the irregular readings.

                         Micronucleus Satellite Report
                                   Comments
                                Action/Response
                                    Page #
Peer Reviewer - Schlesinger

In the statistical section, it is noted that the results of the trend test with Group 4 excluded was not significant, and this was likely due to the low power of the test with available data.  If power is low, then why use the test at all?

Because of the equivocal nature of the data from the first set of slides, a second set of slides was evaluated and appropriate statistical evaluations performed.

The use of both p> designations depending upon the phrasing of the statement adds a certain level of potential confusion for the reader.  All statements should be stated as p< for consistency and to avoid any confusion or misreading of the results.

The use of <p only is the convention in Draft 2

Table 1  -  There should be some error terms (SE or SD) provided with the group means for the % and incidence datasets.  This may help in understanding why a value of 2.7 in the incidence column of the combined gender table is not significantly different from control when a value of 2.4 is.

Error terms have been included in Draft 2 for the first and second set of data presented

It is also unnecessary to indicate that p<0.001 is highly significant.  The p values speak for themselves.

Acknowledged

Peer Reviewer  -  Goldsworthy

Study was properly conducted and scientifically sound; no study deviations were noted.  Positive and negative controls performed as expected and within historical range.  Gasoline-ETBE did not cause any increases in the incidence of micronucleated mature erythrocytes and did not cause any decreases in the proportion of immature erythrocytes.  Group mean values from both the intermediate and high exposure level treatment groups exhibited some increase over negative control values and were outside the group mean historical control range.  Various statistical analyses on pooled data from both sexes and from males and females separately exhibited some significant increases.  Changes were, however, not of high magnitude nor consistently exposure concentration related.  Thus, I concur with the report that Gasoline ETBE Vapor Condensate may cause an increase in frequency of micronuclei in immature erythrocytes.  No evidence of bone marrow toxicity was observed.

A second set of slides from the micronucleus assay was read and the entire report revised to reflect the additional data.  Although there was an apparent increase in group mean micronucleated immature erythrocytes (mie) with increasing concentrations in the second set of slides, none of the values from animals exposed to BG+ETBE Vapor condensate were statistically significant compared to negative controls.  The report has been re-written as draft 2 to include these data.

Editorial:
Page 8, replace "gasoline-TAME" with "Gasoline-ETBE"

Report corrected.
Page 9
211 (b) Research Group Reviewer

The initial draft report [June 20, 2002] gave equivocal results in which only mid-dose female rats showed a statistically significant increase in micronucleated immature erythrocytes (mie) when analyzed by the permutation test; no significant effects were seen in males or for combined sexes.  A linear-by-linear trend test was significant for combined sexes at 20000mg/m[3] even though the mie incidence was lower than at 10000mg/m[3]  -  this significant trend disappeared when the high dose was excluded in the step-down analysis.  No significant trends were seen for males or females evaluated separately.

The second set of slides from the same animals was evaluated and these data along with the original set comprise the second draft report discussed here.  

A second set of slides from the micronucleus assay was read and the entire report revised to reflect the additional data.  Although there was an apparent increase in group mean micronucleated immature erythrocytes (mie) with increasing concentrations in the second set of slides, none of the values from animals exposed to BG+ETBE Vapor condensate were statistically significant compared to negative controls.  The report has been re-written as draft 2 to include these data

General comments from peer reviewers on first report:  Dr Schlesinger requested the addition of error terms [SE or SD] to data sets and also requested that only p< terms be used in discussion and p> terms be deleted for clarity.

p. 8, Summary:  5th paragraph  -  Test material is Gasoline ETBE, not Gasoline TAME.

Corrections have been made.

Corrected.

Please move paragraph 7 to above paragraph 6 and rewrite slightly as:

	"In slide set 2, although there was an apparent increase in the group mean mie with increasing concentration.......etc...  The statistical significance seen in slide set 1 data was not reproduced in slide set 2."   

Revised.

The next paragraph is the explanation for concluding the results of slide set 1 and combined 1 &2 results as not biologically significant.  To the 3[r][d] item in this paragraph add, "The trend test for combined sexes was significant...

Revised.

p. 17, Results:
Tables:  Rearrange the tables so that the table of combined sets 1 & 2, currently Table 1 becomes Table 5 to coincide with the order of discussion in the results.  The combined analyses are not discussed until p. 19.  Thus, Slide set 1 Statistical analysis becomes Table 1, Individual animal data for slide set 1 becomes Table 2; 

Slide set 2 Statistical analysis becomes Table 3 and individual animal data for slide set 2 becomes Table 4.

Revised.

p.17,  1st paragraph:  Change table designations.  Individual animal data is Table 2, not 3

4[th] paragraph: Change table designations.  Statistical analysis is Table 1, not 2.
7[th] paragraph, [Permutation or Wilcoxon Test]: Doses units are mg/m[3], not ppm.  Thus 10000mg/m[3], not 10000ppm

Revised.

Revised.

p 18,  1st paragraph:  Change table designations.  Individual animal data is Table 4, not 5

4th paragraph:  Change table designations.  Statistical analysis is Table 3, not 4.

2nd paragraph: doses are reported as mg/m[3], not ppm.

Revised.

Revised.

Revised.

p.19, 2nd and 6th paragraphs:  doses are reported as mg/m3, not ppm

3rd paragraph:   Statistical analysis table change to Table 5, not 1.

Revised.

Revised.

p.19, 2nd and 6th paragraphs:  doses are reported as mg/m3, not ppm

3rd paragraph:   Statistical analysis table change to Table 5, not 1.

Revised.

Revised.

p.20, 1st paragraph :  Change 10000ppm to 10000mg/m[3]

2nd paragraph:  Please add "A significant linear trend was not seen when sexes were analyzed separately".

5th paragraph describes the results of slide set 2, doesn't it?  Shouldn't slide set 2 be added to the sentence?  Suggest that this paragraph be moved to 3rd paragraph position before the discussion of "One male animal from Slide set 2 showed an individual value (7) outside the historical control range."

In the paragraph describing the reasons for no biological significance, item 3; add "The trend test for combined sexes was significant for Slide set 1....etc.."

Revised.

Revised.

Revised.

Revised.

                  Sister Chromatid Exchange Satellite Report
                                   Comments
                                Action/Response
                                    Page #
Peer Reviewer - Schlesinger

Methods
The statistical analysis used is not adequately described.  The Dunnett's test is a post-hoc test that usually follows an Analysis of Variance procedure.  This is not indicated in the report.

At the request of EPA, a trend test has been added to SCE data analysis.  ANOVA was performed prior to the application of Dunnett's test.  Revisions appear in Draft 2.

Furthermore, the paragraph describing the criteria for a positive conclusion is not clear.  The investigators considered that a test substance was positive if an "...exposure-level responsive and statistically significant increase is observed over a minimum of two exposure levels."  Firstly, how was the relationship between exposure concentra-tion and response assessed?  There is no indica-tion of any statistical analysis for any trend.

Secondly, it is not necessarily valid to conclude that the lack of an exposure level related trend indicates that results should be noted as equivocal.  There can be various reasons why effects at different levels could be similar in magnitude but biologically "real".

Where responses at 2 sequential doses are similar and statistically significant suggesting a threshold for expression, the investigator would likely judge the response to be positive.  However, if positive doses are widely separated and/or values are higher at the lower dose in the absence of toxicity, then the study could be considered equivocal.  The SCE assay was developed as an indicator of DNA perturbation with possible application for identifying potential for chromosome or other genetic damage.  Criteria for positive responses developed by many investigators evaluating numerous assays were designed to be sufficiently rigorous to minimize false positive and negative results in relation to other in vivo genetic assays and to strengthen predictability and reliability.  The designation of "suspect" when statistical significance was observed only at the highest dose, and "equivocal" when positive results are observed at two doses in the absence of an exposure response trend contributed to improving this reliability by ranking the relevance of responses.

Results & Discussion
The actual results are presented at the end of the last paragraph of this section

There is no discussion.

No comments

Furthermore, the 1[st] paragraph of Results should be in the introduction while all of the other paragraphs until the last half of the last paragraph in Results should be in the Methods section. 
Comment has been noted but to be consistent with other reports in this test program no changes have been made.

Peer Reviewer  -  Goldsworthy

This study was properly conducted and scientifically sound.  No protocol deviations were noted.  The positive controls fulfilled the requirements for a valid test.  The report correctly concludes that Gasoline ETBE Vapor Condensate was negative for the induction of SCEs in rat peripheral lymphocytes.

No comments

Comments include:
(1)  Study title should include "Gasoline ETBE Vapor Condensate".

Comment has been noted but to be consistent with other reports in this test program no changes have been made.

(2)  Suggest noting in the Summary Section that the positive control fulfilled the requirements for a valid test.

Statement has been added.

(3)  In data discussion, it would be useful to comment on the observed decrease in mitotic index in top exposure group and whether it has any effect on assay results or interpretation.

Author has considered this difference of no effect and has not revised the discussion.

211 (b) Research Group Reviewer

p. 9, Materials and Methods:
Cell culture and Collection of Metaphases.  On my copy [and apparently on QA copy as well, item #54, Audit 1/13/03], the last sentence describing colcemid addition is missing.  It should read "At approximately 68 hours, 0.2ug/ml colcemid was added to each flask and incubated for approximately 4 hours."  Please check the master copy to be sure the sentence is included.

Added to report.

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

Added to report.

p. 12, Results, 4th paragraph:  Revise sentence describing Dunnett's test to reflect one-tail analysis and trend test.  Adjust sentence in Summary referring to use of Dunnett's test as well.

EPA revised statistics did not alter negative results for BG/ETBE

Added to report.

Agreed.

p. 19, Table 6:  Please check mean SCEs per cell value for rat #5052.  QA indicated it should be 22.2, not 21.7 [item 57, Audit dated 1/13/03].

Report corrected.

p. 20, Table 7:  Please check AGT per group value for Air Control. QA indicated it should be 26.6 (rounded to 27), not 26 [item 58, Audit dated 1/13/03].

Report corrected.

                        Immunotoxicity Satellite Report
                                   Comments
                                Action/Response
                                    Page #
Peer Reviewer - Schlesinger

Immunological Evaluations
Tables 1 & 2  -  Is it necessary to repeat the methods at the bottom of each table.  All this previously indicated information makes it hard to find the key to the various abbreviations used in the table.

While these footnotes are unnecessary in the context of the report, Dr White prefers to include them because they allow the tables to stand alone.

Peer Reviewer  -  Goldsworthy

This study evaluated the potential of four weeks of exposure to Gasoline-ETBE to affect the humoral immune component of the immune system when evaluated by antibody-forming cell response to T-dependent antigen sheep erythrocytes.  This study was conducted properly and was scientifically sound.  Data demonstrate that Gasoline-ETBE did not affect terminal body weights, spleen or thymus weight.  Gasoline-ETBE vapor condensate did result in a dose-related decrease in the spleen IgM antibody-forming cell response to sheep erythrocytes.  Decreases in AFC/spleen were also seen in the two highest exposure levels.  The positive control CPS resulted in the anticipated results.

The conclusion that, under the experimental conditions of this study, Gasoline-ETBE did adversely affect the functional ability of the humoral immune component of the immune system accurately reflects the study results.  No effects were noted in any exposure group on body weight, spleen weight, thymus weight, or spleen cell number.

No comments needed

211 (b) Research Group Reviewer

 Appendix B, Sponsors Exposure and Animal Data:  According to QA [items 59, 60] body wt, body wt gain, food consumption and resulting means tables have animals mixed among groups.  I assume this did not occur in Appendix A. Individual Animal Sacrifice Data used by Dr. White in the report.  Please resolve.

p.9, Table ES-1; As a stand-alone table, presentation of maximum effect at 10000mg/m3 could be misinterpreted as indicating there was not significant effect at 20000mg/m[3]. Consider adding a footnote containing the statement that 20000mg/m[3] is statistically significant data even though it is slightly lower than 10000mg/m[3].

Issue rectified by HLS IT and tables re-run.

Footnote added as requested.

p. 11, Experimental Design, 1st paragraph, line 10:  Handling of cyclophosphamide is described twice.  The sentence beginning "Cyclophosphamide was dissolved...." can probably be deleted.

Deleted.

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

Paragraph has been moved.

Please provide a rationale for why only females were used in this assay.  Peer reviewers have requested this clarification in other immunotoxicity studies in this series.

The immunotoxicology study is the only assay that used only female rats.  The WG accepted the recommendation of Dr. Kimber White that females were the appropriate sex for this assay since in general they provide a more robust immune response than males.

p.15,  In earlier studies, EPA inquired whether viabilities of 84% were considered low or correlated favorably with historical values.  Could historical data from Dr. White's laboratory be provided to address this inquiry?  Although not requested in the BG/ETBE review, adding this information would make the report consistent with the others in the series.

A paragraph discussing viability has been inserted on page 15.

p. 16.  Functional results: Dr. White indicated that the decrease in the IgM-antibody-forming cell response was dose-related.  Although effects were induced by two doses of BG/ETBA, the response was not technically dose-related because the higher response was seen at the lower dose rather than at the highest dose.  Please delete dose-related or revise the sentence.  [also cited in EPA review, Aug 26, 2003, and QA item 64 Audit 1/13/05].
Dose-related was deleted from the discussion.

                             GFAP Satellite Report
                                   Comments
                                Action/Response
                                    Page #
EPA 

GFAP
We have reviewed the API report on the subchronic study for Gas+ETBE vapor condensate effects on GFAP submitted by Dr. O'Callaghan.  We agree with the view of Dr. Callaghan that the statistically significant results in male rats are small and not dose-related (and in fact show a decrease in GFAP).  The significance of a decrease in GFAP of that magnitude in regard to neurotoxic damage is unclear and probably represents technical variations in the assay.  Likewise, the small, non-dose related increase in GFA in the female striatum and the rest of brain is unlikely to represent neurotoxic damage.  We noted larger increases in GFAP levels in the hypothalamus (especially female) that were not significant.  Careful evaluation of the raw data indicated one data point of five that was unusually high, and we would suggest that this is a dissection artifact.  Overall, we agree with the peer review comments and the API response

No comment needed.

Peer Reviewer - Schlesinger

p. 17. Statistics.  Which post hoc test was used in this analysis and was there any test for homogeneity of variance?

The statistical methods used in this assay were analysis of variance followed by post hoc comparison of treatment means (JMP[(R)], SAS) with significance established at p<0.05. (p. 17 of report).

In addition, the notations in the text of number of male and female groups with changes seem to be in error.  Two male groups rather than three showed effects, while one female rather than two showed effects.  I think that the author confused number of exposure groups with number of tissue groups.

Both the reviewer and the investigator are correct.  In general, all subsets of data within a dose group are considered part of that group; thus, statistically significant decreases in GFAP were seen in the cortex region and the rest of brain sample in low dose male animals (Group II), and in the cortex in high dose males (Group IV).  In low dose  (Group II) females, increases in GFAP were observed in the striatum and in the rest of brain sample.  However, Dr. O'Callaghan characterizes each brain region at each exposure level as a group, increasing the total number of groups considered.

Peer Reviewer  -  Goldsworthy

These studies appear to be properly conducted.  Editorial comments from previous reviews regarding the format, i.e. lack of study specifics, remain.  Pituitary GFAP level could only be detected in one male one female rat; due to the paucity of data and inability to make any conclusions from this region of the brain, this data should be omitted from the tables and noted in the text that analysis of pituitary could not be adequately performed.

The pituitary data have been excluded from Tables 5 and 6 although the data from 1 male and 1 female are included in Table 1, which lists individual animals.  Text has been revised appropriately.

211 (b) Research Group Reviewer

p.8, Introduction: first full sentence at top of page  -  Change  text as follows "Inhalation exposures to gasoline and gasoline plus each of the 6 fuel additives have been performed (replaces `will be undertaken").  Also, correct "with a two generation reproduction toxicity study that includes a neurotoxicity component" by adding `for gasoline and gasoline plus MTBE vapor condensates only."

Report corrected.

Although it has been decided by the Research group to maintain the GFAP reports in the current format, some general suggestions to make the report more consistent with other satellite studies are presented.

No comment needed.

p. 14-16,  V. Commentary  A. Background information:  This section belongs in the Introduction and can be integrated with the second paragraph there beginning " A universal cellular reaction......"

Report format has been accepted as is.

p.17, D. Typical Results:  Figure 2 displays "typical GFAP assay values from different regions of rat brain."  I assume these values are historical data from untreated control rats in Dr. O'Callaghan's laboratory.  Could the number of rats evaluated, strain, sex and age from which these data were collected be provided, perhaps in a footnote?  Do these variables affect the regional differences in GFAP values within a species?  Similar comments also made by Peer reviewers and QA.

Changed first sentence to: "Typical GFAP assay values obtained at this laboratory for different regions of the rat brain are presented in Fig. 2 (i.e. historical data)."  And a citation for these results is now mentioned in the Figure Legend (p. 21)  and reference section (Martin and O'Callaghan, 1995).

Results and Conclusions:  
In the 3rd sentence, the Figure designation for levels of GFAP across different brain regions is incorrect.  It should be Fig. 2, not Fig. 1.

Figure designation has been corrected

Delete sentence concerning " A recently developed fluorescence based GFAP ELISA assay for pituitary data....".  Although all of this information is of interest, it is appropriate in the discussion section of a publication, not in a study report for regulatory submission.  These changes have already been made in the 2[nd] draft of the gasoline GFAP report.

Section has been deleted.

Pituitary data:  I am still not sure whether the pituitary data belongs in any of the oxygenate studies.  Data is sketchy at best and the assay may not detect anything of value in this organ.   

Table 7:  There are virtually no detectable GFAP data in any dose group except for 1 male and 1 female at the 20000mg/m3 level.  The individual values for these animals are much higher than seen in any other BG/oxygenate study where pituitary numbers, if present, are 0.02-0.08, not 8.08 or 6.4.  Please check these values against raw data [QA says these numbers are incorrect, item #74, Audit 1/13/03].  These data should not be included in Tables5 & 6, which summarize mean values.  An average value cannot be obtained from only one data point and there are no pituitary control values.  [Pituitary data issues are also cited by Peer reviewers and QA]. 
The pituitary data have been excluded from Tables 5 and 6 although the data from 1 male and 1 female are included in Table 7, which lists individual animals.  Text has been revised appropriately.