### 211(b) Toxicology Research Group ETBE One-Generation Reproducti on Toxicity Study Report

Reviewer Checklist

1 5/4/2010 

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EPA
In general, the NHEERL reviewers concluded that the

studies were conducted in accordance with the testing guidelines, once changes detailed in the API peer review response are made. Additional comment is requested on the following points: The peer reviewers were concerned that statistically significant increases in testis, seminal vesicle, and ovarian weights relative to total body weight in the high exposure group were considered spurious. It appears that this poor

correlation was related to the significant weight loss reported in the parental animals in the 20,000 mg/m 3 exposure group, since the application of covari ance analysis eliminated the earlier statistical significance of the individual organ weights. We would request that the study group perform a

statistical evaluation to confirm that the observed 20% increase in seminal vesicle weight (relative to body weight) does not reach significance when compared to brain weight. Additional discussion added to end of section

3.4.2. Ok pp 41-42 OK



### Peer Reviewer – Schlesinger
The study was conducted in a scientifically sound manner and followed the appropriate protocols. There were no significant deviations from these protocols that would have

affected the outcome of the st udy. However, there does seem to be some indication that the 2,000 mg/m 3 exposure level is not a NOAEL, as noted below. Dr. Schlesinger’s comment concerning the NOAEL = 2,000 mg/m 3 seems to refer to lower absolute body weights in low dose female rats premating. However, the effect was statistically significant in the 2000mg/m 3
females at Day 63 (p<0.05) only; on Day 69, the average female body weight in the low dose group was within 10g of control value compared to weights lower by 17 and 19g respectively in the 10000 and 20000mg/m 3
groups. In addition Table 6 demonstrated that over the entire period, body weight gain was not significantly lower in 2000mg/m 3 females. The RG agrees with the study director’s evaluation of the absence of biological significance for decreased body wt at the low

dose. Ok

Specific comments are as follows: There should be consistency in the Group numbers. In some places they are noted as I, 11, III, and IV, while in others they are noted as 1,2, 3, and 4. This inconsistency has been brought to the attention of the study director and, where practical, adjustments have been made. However, in computer generated tables and compilation of raw data the number format is predetermined. This observation does not affect the validity of the data Ok

p. 29 Statistical Analysis: 2.15.1. It is not clear why a one-way ANOVA was performed at each time interval, resulting in the need to perform multiple ANOVAs. Is there any reason why two- way ANOVA was not used? This would have allowed evaluation of exposure groups at all times for each en dpoint. This could be followed by an appropriate multiple comparison test. The computerized statistical testing package employed at the laboratory is designed to perform one-way A NOVA at each time interval and is used for a range of multiple

dose studies. Ok

### 211(b) Toxicology Research Group ETBE One-Generation Reproducti on Toxicity Study Report

Reviewer Checklist

2 5/4/2010 

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What is the rationale for having a separate discussion of the method of analysis of the organ weight data when these data also used the ANOVA procedure? Furthermore, if Bartlett's test showed there to be unequal variances, why was a parametric test still used? Organ weight statistics were discussed separately because, unlike other parameters,

Bartlett’s test was used here to assess variance. Parametric tests were used onlywith homogeneous data. If data were nonhomogeneous, the non-parametric Cochran &

Cox’s modified t-test was used to evaluate significance. RG will discuss rewording of the first part of this paragraph for clarity with the study director.

We have reviewed and not made any changes

because:

1. 1 st paragraph describes TASC procedures (all parameters except parental organ weights).

2. 2 nd paragraph describes Xybion procedures (parental organ weights). Xybion’s stats are wholly parametric (no non-parametric), so the

statement about Cochran & Cox above is not

correct.
If the 2 nd paragraph refers only to Xybionstatistics why is Cochran and Cox modified Ttest mentioned in that paragraph (see p. 30). If entry is incorrect should it be deleted from text or explained differently? Text is correct per Xybion stat package

3. 3 rd paragraph describes Startox procedures (analysis of co-variance for selected organs). Ok

Provide the rationale for the ANOVA procedure with certain organ weights using terminal body weight as a single covariant. Which organ weights were evaluated using this procedure? Absolute organ weights that fall within normal range may become significantly different from controls in the context of terminal body weights that are lower or higher than controls. Where significance in relative organ/body wt seems skewed due to body weight variation, the analysis of covariance is used to assess the statistical significance of these values.

Organs evaluated by covariance were epididymis, seminal vesicle, testes and ovary weights in the 20000mg/m 3 group to determine if statistically significant increases relative to body weight identified by Dunnett’s test were accurate. Analysis of

covariance demonstrated that the earlier statistical significance was spurious for all organs. In addition there were no statistically significant increases in absolute organ weights or organ weights relative to brain weight. Ok

Why were tests conducted at both the 1% and 5% level of significance? The tests and level to be used should be determined a priori before the study begins and only one such level of significance should be used. Both 1% and 5% levels of significance were pre-selected since they were inherent in the laboratory’s computer statistical package. Comparison at two levels of significance has been common practice in this laboratory. ok

### 211(b) Toxicology Research Group ETBE One-Generation Reproducti on Toxicity Study Report

Reviewer Checklist

3 5/4/2010 

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It is noted that, "Dose groups were eliminated from statistical analysis if their standard deviation was 0 and/ or N...in the group was less than or equal to two." How many such cases were encountered in the study? This is a standard statement for the statistical methods section. In this study, Table 1 Summary of Survival and Pregnancy provides examples of where the n is 0 or 1 and this statement is relevant. For example, No animals died pre-mating in controls and high dose groups but one each died in the low and mid dose groups. No statistical analysis was applied. Ok

p. 35 Chamber Monitoring: 3.1.

It is noted that there were some differences between the

measured and the nominal concentr ations in that they varied from the expected ratio. Th e explanation for this was suggested to be a combination of slight inaccuracies in the

calibration of the chamber airflows and the calibration of the

IR monitor. This reviewer f eels that there should be no inaccuracies in either of these, especially in the calibration of the monitor. The investigators need to assure that the

calibration of all monitors an d flows are extremely accurate.

There should be no such in accuracy of any atmospheric monitor. In any case, the correspondence between the measured and nominal levels is very good, so it is not clear why any explanation in this regard is warranted. To be precise, the report stated that the ratio of

the measured to nominal concentration was not 1:1, and speculated that the cause MAY have been due to calibration error. In any event, the inaccuracies are believed to be slight. The RG feels confident that the reported chamber concentrations accurately reflect the atmospheres to which the animals were exposed. HLS – will you want to delete the calibration error explanation

Yes and deleted. OK The investigators need to explain the source of the particles in the exposure atmospheres. Was the incoming chamber air filtered appropriately? Any particles could ad as adsorption sites for vapors in the exposure atmosphere. With regard to the presence of particles, the RG notes that the mean particle concentrations were comparable between the control and treatment group chambers. The primary purpose of making these measurements in a study of this nature (high vapor concentrations) is to make certain that vapor condensation is not occurring and to preclude the possibility of aerosol formation. The results provided in the report confirm that gasoline exposure was to vapors and not aerosols. Some particles are invariably present in chamber atmospheres, representing background levels. In fact, careful analysis of the data in the table on page 36 indicates that the particle concentrations in all of the chambers including controls ranged from about 7 to 9 micrograms/m 3 per cubic meter of air. For comparison, it has been reported in

EPA's PM 10 Criteria document that annual background PM 10 levels in eastern cities range from 5 to 11 micrograms / cubic meter of air. Thus, the RG believes that the particles in the chambers reflect ambient background values. ok

p. 37 Bodv Weight: 3.2.3. The investigators state that the only exposure related effects

were in males and females at the highest concentration, and females at 10,000 mg/m 3 . However, data in Table 5 show Overall gasoline +ETBE vapor exposed animals showed slight to significant lower absolute body weights during pre-mating

. ok

### 211(b) Toxicology Research Group ETBE One-Generation Reproducti on Toxicity Study Report

Reviewer Checklist

4 5/4/2010 

### Comments Action/Response 

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significant difference at Day 63 in females exposed at 2,000 mg/m 3 and a trend upward in the same exposure group at Day 69. Thus, it does appear that, with increasing time of exposure, there is a real effect on this parameter in the lowest exposure level group. However, the effect was statistically significant in the 2000mg/m 3 females at Day 63 (p<0.05) only; on Day 69, the average female body weight in the low dose group was within 10g of control value compared to weights lower by 17 and 19g respectively in the 10000 and 20000mg/m 3 groups. In

addition Table 6 demonstrated that over the entire period, body weight gain was not significantly lower in 2000mg/m 3 females. The RG agrees with the study director’s evaluation of the absence of biological significance for decreased body wt at the low

dose. p. 38 Maternal Body Weight: 3.2.4. Data in Table 7 and 9 indicate what appears to be an early decrease in weight gain at 20,000 mg/m 3 . Furthermore,

clarification is needed concer ning the last sentence in this section regarding differences in the premating period. Table 7 and 9 show absolute body weights throughout gestation and lactation, respectively, not weight gain. Wt gain data during gestation is provided on Table 8, and there are no statistically significant differences in wt gain during this period. Although the high dose females showed significantly lower absolute weights during gestation and lactation, they started gestation weighing less than controls due to low pre-mating weight. The weight gains during gestation and lactation were comparable to controls. The RG accepts the evaluation of the study director. No changes in text have been made. Ok

p. 40. Postmortem Data: 3.4.1. Data in Table 19 appear to indicate a dose-related trend of increasing lung discoloration. The incidence of lung discoloration in each dose group (sexes combined) does show a general increasing trend in the mid and high dose groups [control 9.8%; low 8.2%; mid 17.6% and high 16.7%]. The report text (p. 40) indicates that lung discoloration was noted but in the absence of any effect on lung wt or histopathology was not considered treatment

related.” Occurrence of discolored foci is considered a background finding which may occur non-specifically with a variety of inhaled materials, and sometimes is correlated with histologic identification of alveolar macrophage aggregates. In this study, the pathologist did not report any remarkable changes in lungs of treat ed animals to support a significant correlation with grossly observed lung discoloration. The RG accepts the evaluation of the study director. No changes

in text will be made OK

p. 41. Organ Weight: 3.4.2. The investigators note that th ere is an effect on kidney weights at all exposure levels in the males. Table 21 seems Table 21 evaluates relative organ/body weight data. Two statistical methods are used, one Ok

### 211(b) Toxicology Research Group ETBE One-Generation Reproducti on Toxicity Study Report

Reviewer Checklist

5 5/4/2010 

### Comments Action/Response 

### Page
to indicate such an effect only at the two highest levels of

exposure. for homogeneous data and one for nonhomogeneous data. The statistical significance at the two highest doses was identified using Dunnett’s test, and the significance at 2000mg/m 3 was determined using the Modified t-test for non-homogeneous data. The symbols used to designate significance are different depending on the test employed (see footnote on Table 21). It is not clear whether the lack of any histopathologic correlation with the change in liver weight is meant to suggest that the noted effect on weight is of no biological significance. As pointed out by Dr. Goldsworthy, the statement on p. 41 indicating the absence of histological changes in livers is incorrect. Table 1 in Methods (p.2 6) indicates that no microscopic evaluation was performed on the livers of any animals. This is confirmed by the RG review of Appendix Z, Individual Animal Histopathology, in which “no micropathologic observations on tissue” was listed whenever necropsy data on livers was cited in any group. The study director will

correct this statement.

Statement has been corrected and considered in the NOAEL of 2000mg/m 3

. OK Concerning the biological significance of the increased liver weight in mid and high dose male rats, the effect was considered significant enough to be used as a factor in setting the parental NOAEL at 2000mg/m 3

.

What is the basis for the conclusion that changes in epididymis, seminal vesicle and ovary weight are considered to be spurious? See response on top of p. 2 addressing “Use of analysis of variance for certain organs using terminal body weight as a single covariant. Discussion has also been expanded on pp 41-

42

. Ok

Table 21

Statistical results are indicated for two different tests. Why were two tests on the same dataset, and is the reader to accept a conclusion of significan ce if either test indicates this? Some rationale for this type unorthodox statistical procedure needs to be provided. The statistical routine is a conditional regimen with different tests required for data sets depending on whether they are homogeneous [ Dunnett’s test] or non-homogeneous [Modified T-test]. This is typical for statistical evaluations of toxicology data. The

reader should never assume that statistical significance with either test equates to biological significance without reviewing the data set carefully including the pattern of response, the direction of response, the degree of response, etc. Ok



### Peer Reviewer – Goldsworthy
The study was scientifically sound and was properly conducted. Exposures were prop erly conducted and adequate toe test the potential reproductive toxicity of Gasoline ETBE Vapor Condensate. None of the protocol deviations noted No response needed

### 211(b) Toxicology Research Group ETBE One-Generation Reproducti on Toxicity Study Report

Reviewer Checklist

6 5/4/2010 

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would be expected to adversel y affect study results, data integrity or conclusions.

No effect of treatment was noted on survival, clinical observations, feed consumption, estrus cycles, mating, fertility or gestation indices, or no other reproductive parameters up to parturition. An exposure-related decrease in body weight gain was observed in high dosed male andfemale rats, and the mid-dosed female rats during the premating interval. Text (on page 37) notes the decrease in body weights in female rats exposed to the 10,000mg/m 3
gasoline-ETBE vapor condensate; however, neither the summary (pages 5-7) nor the conclusion (page 43) note this adverse finding. This should be noted in both the summary

and the conclusion. No treatme nt differences were observed with pregnancy rates, duration of gestation and percent of females completing delivery. No differences between treated and control groups were noted in all parturition parameters, including total number of pups delivered, viability and lactation indices, the number of implantation sites per litter, the pup sex ratio, and the number of live pups per litter. Additional sperm count, motility and morphology was not altered by the treatment. Dr. Goldsworthy’s comm ent is correct. The Conclusions and Summary will be revised to include decreases in body weight for 10,000mg/m 3 females and cite the criteria in setting the NOAEL where appropriate.

Done.

Checked both locatio ns Summary entry OK Conclusion p. 43 As presented in Summary, add “females only” in parentheses after 10000 to read “ decreased body weight gain in the 10000 (females only) and 20000mg/m 3
exposed parental animals”

Done ok

There were no exposure-related differences in macroscopic postmortem assessments. Increased kidney weights were observed in male rats (parental generation) at all exposure levels; this is consistent with the expected induction of hydrocarbon necropathy. Microscopic evaluation was not performed to confirm this observation. Liver weights were increased at the two higher exposure levels in male rats. Text on page 41 notes that this alteration "were not correlated with any histopathol ogical finding". However, the tissue list on page 26 notes that liver was not examined microscopically; thus text should be modified. It is not clear why, in this study, brains, kidneys, liver and spleen were not examined microscopically when they were examined in the gasoline-DIPE one generation reproductive study. There were statistical differences noted in a number of organ weights in treatment groups, especially the high dose group; these differences, which included alterations in epididymides, testis, ovaries, seminal vesicle and spleen weights, were considered not a ttributed to the test article

because of the absolute differ ences noted, the lack of a treatment-related patter, and the lack of correlation to body weights. Whereas it may be correct to not attribute these changes to treatment, these di fferences are significant innature to note in summary section. There were no exposurerelated difference in body weights, macroscopic postmortem evaluations and organ weights in lactating pups from Air

Control animals. Liver weights: This statement will be corrected. See response above to Dr. Schlesinger comments (p. 4, second paragraph under Organ wts).

Brain, kidney, liver and spleen were not examined histopatholo gically because it was decided by the RG that sufficient histopathology data on these organs was available from the 90-day BG+ETBE study in which no significant effects were observed, and by read-across from other gasoline+ oxygenate blend studies. In actuality, the histopathology performed in the DIPE 1- generation reproduction study was a deviation from the standard protocol for the 1- generation studies.

The statistically significa nt increase in relative weights of epididymis, testes, ovaries and seminal vesicles at 20000mg/m 3 , were also cited by Dr. Schlesinger. Dr Goldsworthy did not disagree with the conclusion that the significance of these changes as treatment induced events was spurious, but as he did

recommend that these effects be included in the summary section with appropriate explanation, the RG requests the study director to provide a brief comment on thesefindings indicating they are not treatmentrelated events

### 211(b) Toxicology Research Group ETBE One-Generation Reproducti on Toxicity Study Report

Reviewer Checklist

7 5/4/2010 

### Comments Action/Response 

### Page

Additional discussion added to end of section

3.4.2. OK

In summary, test animals were properly exposed to adequately test the potential reproductive toxicity of gasoline-ETBE. A NOAEL of 2,000 mg/m 3 was determined. This NOEL setting is based on decreased body weight gains and increased liver weights in parental animals in the 10,000 mg/m 3 and 20,000 mg/m 3 groups [note text in summary and conclusion sections do not mention the decreased body weight gain with regard to the NOAEL setting]. The NOAEL setting of 2,000 mg/m 3 does not take into account the increased renal weights observed at all exposure levels; this finding is consistent with hydrocarbon nephropathy observed in sim ilar animals exposed to test substance and generally regarded as not to be relevant to human risk assessment. There was no effect at any exposure level on reproductive performance, including mating, fertility, parturition, lactation, and offspring survival. Thus, with respect to reproductio n performance, a NOAEL of 20,000 mg/m 3 was appropriately concluded. Dr. Goldsworthy pointed out that the parental NOAEL of 2000mg/m 3 did not take into account the renal changes in male rat and perhaps this should be stated.

HLS please include a statement in the Conclusion and Summary that the NOAEL

was determined exclusive of the renal effects due to light hydrocarbon nephropathy seen in male rats, since these effects are not relevant

to humans.

We have been consistently stating in all of the 211(b) reports that the male renal effects are not considered in the NOAEL setting. OK



### 211 (b) Research Group Reviewer

General Comments: The report should have sequential numbering from start to

finish. This will be done at report finalization. OK

The numbers for cannibalized/stillborn/missing/found dead are confusing; please see comments related to T. 20. No change to report. OK as is per Robert

Parker’s review. OK Historical control data is listed with released pups as part of App. A, but also appears in the Table of Contents as App.

DD. TOC corrected. OK

However, App. DD is the Protocol and Amendments, and the subsequent appendices are also in error. Please straighten this up. TOC corrected. OK

Specific Comments: P. 5, Summary, 4th paragraph Please consider changing th e word, "reasonably" to"acceptably," since there should be an SOP or protocolspecific criteria for concordan ce of nominal and analytical concentrations. The same change should be made later in the same paragraph on p. 6. Revised and also in the R&D. OK

Also, please consider deleting the sentence, "The exact cause of the differences ....." as unnecessary. Deleted. OK

P. 6, 2nd paragraph Female premating body weight gain was also decreased at 10,000 mg/m3 (p. 37). Revised. OK

P. 6, 3rd paragraph: Please spell "2". Done P. 7, 3rd paragraph Again, female premating body weight gain was also decreased at 10,000 mg/m3. Revised. OK

P. 16, Dispensing

Chevron Research and Technol ogy Company. The supplier Revised to Chevron Global Technology Services Company (per other reports). OK

### 211(b) Toxicology Research Group ETBE One-Generation Reproducti on Toxicity Study Report

Reviewer Checklist

8 5/4/2010 

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can be described either as Chevron Global Technology Company, which is the name on the contract, or as Chevron Research and Technology Co mpany, that provided the sample, whichever QA considers to be most appropriate. P. 22, Estrous Cycling Please note the study days for the start of the daily vaginal

smears. Added. OK

P. 23, 2.12.4, Litter Observations Please make note of the lung floatation test to distinguish stillborn and found dead pups, also that autolyzed remains found on the day of parturition were presumed to be

stillborn. Added. OK

P. 32, Protocol Deviation 1 Please note the sex and exposure group of the two animals not exposed on Day 97. Added. OK

P. 33, Protocol Deviation 8 Thank you for describing the incident clearly and thoroughly. Could you also please add the phase of the lactation period when the incident occurred? I.e., most or all of the pups were LD > 25. Added. OK LD was 25-27.

P. 35, final paragraph Please consider changing th e word "reasonably" to "acceptably" as requested above. Also, please consider deleting the sentence, "The exact cause.. ." as unnecessary. Revised. OK

P. 37, Mortality/Physical Observation data The two animals killed accidentally are not described similarly in Table 2. 2073 is listed as found dead. 4553 is

listed as accidental death. Shouldn't these be described in the same manner if the same thing happened to each of them? Revised. OK

Body Weights- Premating Period Please see note below regarding App. H. See response below.

P. 38-39, Estrous Cycle Data The data suggest that pseudopregnancies were included in the group - summaries and comparisons, which shouldn't happen. Please check 1559, a control animal with a single 18 day cycle, appears to be included in the control group summary. This value is sugges tive of an artifactual factor and diminishes the ability to detect intergroup differences. We have reviewed the data and the tabulation and confirm that pseudopregnancies were not included (including for #1559) in the group -

summaries and comparisons. OK

P. 40, Pup Body Weight Data: Please include an explanation for the multiple age ranges for which weight gain was evaluated. Pup weight changes are preselected by the TASC system and there is no way to change which day ranges to include/exclude. OK P. 41, Organ Weights, 2nd paragraph

- Please spell "2".

- Also, liver weights were only affected in males; please

include gender as a qualifier in the first sentence.

- - Done OK

- Done OK

3rd paragraph It's been my understanding th at testes and ovaries aren't usually well-correlated to body weight, and that absolute organ weight is usually the preferred endpoint for their

evaluation. Are there other reasons to consider these statistically significant differences to be spurious that could We have reviewed and confirm that the offered explanation is sufficient. [See expanded discussion on pp 42-43]

### 211(b) Toxicology Research Group ETBE One-Generation Reproducti on Toxicity Study Report

Reviewer Checklist

9 5/4/2010 

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be cited?

P. 43, Conclusion Decreased body weight gains were also noted in the 10K females (p. 37), and so should be noted here as well. Added OK

Tbl. 14 For the control group, please fo otnote that the value for the number of estrous cycles includes one animal with no complete cycle. Please see above, 1;..38-39, regarding concern about the control group cycle length value. Animal was pseudopregnant and reformatted

table excludes this animal. OK

Tbl. 19 I am disappointed that GD 25 animals were terminated without necropsies; please explain. This was done as per protocol amendment#1, point#2. OK

Tbl. 20, p. 1 Please indicate if cannibalized should be interpreted as missing. No: cannibalized means that some tissue was still present. OK

Tbl. 20, p. 2 Lung Floatation Test - Found Dead

The number for the controls shou ld be three, not two. Please

see comment below. Corrected. OK

Tbl. 20, p. 4: Lung Floatation Test - Found Dead App. W, p. 3,

Indicates that #1561/14F should not be listed as a stillborn based upon the results of the lung floatation test and milk in

stomach. Please correct Tbl. 20. Corrected. OK

Tbl. 20, p. 5: Lung Floatation Test – Stillborn The numbers match up if the autolyzed and cannibalized pups are included. This may need footnoting for

clarification. Numbers for Lung Floatation Test – Stillborn match Appendix W. Pups/Litter Evaluated includes cannibalized/autolytic pups for which the floatation test could not be performed. Footnote added to pages 4 & 5. OK App. H. p. 5- 8: Individual body weight for the Day 0-69 interval should be

shown as in Table 6. Added. OK

App. S, p. 1 1561114F is listed as stillborn, but according to App. W individual pup necropsy observations, this pup should be

classified as found dead. Please correct. Corrected. OK

App. U, p. 1

Please make the same co rrection for 156111 4F Corrected. OK

App. AA There are multiple page 1's. This seems like a bad idea and could be confusing. This appendix includes historical control data for pups; should this be in App. DD? Pagination will be corrected in the final report. HCD is in Appendix AA (TOC corrected).

OK App. DD: The historical control data appendix is missing. TOC corrected. OK App. EE - GG: All are mislabel ed according to the Table of

Contents. Please correct. TOC corrected. OK



### 211(b) Research Group QA/QC Reviewer
The following items are co mments or require further

consideration: Compliance Statement The sponsor also needs to sign a compliance statement. It No change. Sponsor signature line is already

### 211(b) Toxicology Research Group ETBE One-Generation Reproducti on Toxicity Study Report

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10 5/4/2010 

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can be a separate one from the Testing Facility’s, but there must be one signed by the sponsor. included on the Compliance Statement. OK

Page 2, Compliance Statement Since it was the sponsor’s responsibility to maintain the method of synthesis, fabrication, or derivation of the test fuel, and this had not been completed, it must be included in the sponsor’s compliance statement. Exception added. OK

Page 4, Quality Assurance Statement: Since HLS has inspected the sp erm evaluation subcontractor (PAI) and is responsible for ensuring their GLP-compliant status, these facility inspections should be listed on the QA statement for this study. Added. OK

Draft Report, Page 3 Should the signature of the reproductive toxicologist who reviewed this report also be added to the signature page? Signature page revised OK

Draft Report, Page 13, Section 2.2.6 Terminal Sacrifice

The dates for sacrifice for the nonpregnant females have not been included (June 22, 23, 25 and July 5). Added OK

Draft Report, Page 19, Section 2.9.8 Bedding Analysis States that analyses for bedding can be found in Appendix CC. Analyses are available, but are from 1999. Are there any more recent/representative analyses? These are the analyses that were received with the lots used on this study. OK

Draft Report, Page 32, Protocol Deviations All adult animals were to receive gross necropsies. Since the non-pregnant females were not given a necropsy and tissues were not saved, this is a deviation and should be included in

this section. Protocol Amendm ent No. 1 indicates they will be euthanized, but doesn’t indi cate a change with respect to necropsies and tissue harvest. Deviation added to report and data OK

Draft Report, Page 38, Section 3.2.4 Maternal Body Weights, Gestation and Lactation Periods The absolute body weights for the females in the highest level were statistically significant during lactation and gestation. Should this fact be discussed in this section? Additional discussion added to this section.

OK

Draft Report, Page 37, Section 3.2.1 Mortality – The raw data records that male rat #2703 was found dead, not “accidentally killed” as the text on Page 37 states. Corrected. OK

Draft Report, Page 42, Section 3.4.3, Released Pup Data, Appendix AA: It may not be accura te to indicate that this Air Control set of pups (releas ed) was comparable to the historical control pups of similar age as in Appendix AA, as it appears that Day 21 values are used for Study 4202, making the mean much lower than it should be. The historical values range from 73 g to 82g, where these values are around 66g. The nonreleased pups are even a little

smaller than the historical control. Were statistics run on these to verify that these anim als were similar to historical

controls? Appendix AA, Study 4202 terminal body weights corrected to Day 28 values. OK

Draft Report, Table 16 – The indices should be calculated to include survival to Day 28. All calculated indices stop with Day 21. No change possible due to system limitations.

OK

Draft Report, Table 16, Groups I and III – The Number of pups dying, missing and/or cannibalized Days 5-21 and 0-21 should be checked. This data also changes the pups surviving Numbers reported on Table 16 appear to be correct and match data in Appendices R and

S. Still cannot verify some values, but they

### 211(b) Toxicology Research Group ETBE One-Generation Reproducti on Toxicity Study Report

Reviewer Checklist

11 5/4/2010 

### Comments Action/Response 

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21 days and the lactation index for these two levels. Appendices S and R do not agree with the Table 16

tabulations. are close. Groups I and III on Table 16 checked against Appendix R and Appendix S. No changes

apparent:

Group I – 1558-11 (Day 9), 1562-2 (Day 15) and 1562-5 (Day 16) = 3 pups dying/missing/cannibalized Days 5-21.

6 pups on Days 0-4 + 3 pups on Days 5-21 = 9 pups Days 0-21.

Group III – 3555-4 (Day 21) = 1 pup dying/missing/cannibalized Days 5-21.

4 pups on Days 0-4 + 1 pup on Days 5-21 = 5 pups Days 0-21. OK Draft Report, Table 17 – The observation of laceration mid abdomen has not been reported (Animal #355-9). Also the observation for tip of tail missing should be 2/2 for Group IV. Animals #4561-2 and 4561-7 both had the tip of the tail missing for one observation. Laceration observation added. ok

Tip of tail observation was seen in 2 pups in 1 litter. Frequency/animal is the number of

times an observation occurs/number of litters

it was seen in. ok Draft Report, Table 18, Summary of Pup Body Weight Gain – This table does not report the weight gain for Day 4-14 and Days 7-21. The only weight gain report from Day 1 was Days 1-4. Should the other time periods also be reported for weight change (Days 1-7, 1-14, 1-21, 1-28)? Not available based on TASC program hardcoded output. Ok

Draft Report, Table 19, Gross Observations Preface: Animal

number 2559 is mentioned twice when I believe number

2553 should be referred to. Pl ease verify. It also indicates

that observations for this an imal are presented in Appendix V. Appendix V is Estrous Cycle determinations and does not contain gross observations. Animal number corrected. ok

Reference to Appendix V removed. ok

Draft Report, Table 20, GROSS EXAM: I cannot verify the “Litter Incidence or Pup Incide nce” values for this table. What do they mean? It is not the litter incidence of dead pups as that would be a much higher value. The table is divided into Pups Died, Pups Stillborn, Pups Culled and Weanlings. Litter incidence and pup incidence are dependent on

these sub-sets. OK For instance, Pups Died in Group 1: Three pups have gross exams (1562-2, 1562-5 and 1573-10). The Litter Incidence for Gross Exams is 2 litters and the Pup Incidence for Gross Exams is 3 pups. Since all three pups were found to be autolytic, the same Litter and Pup Incidences can be found for Autolysis. Draft Report, Table 21, Parental Organ Weights: Should the animals (dams) from the litters on the rack that fell be included with others for terminal body and organ weights? Some of these animals were lactating 2 or 3 days fewer than others. The same question could be raised for Appendix K where lactation day 28 weights are reported for all animals (except not pregnant). Lactation may be at a minimum at this point, but some body weight difference with these animals may be realized. We have reviewed and do not feel it is necessary to make any changes since weaning occurs around LD21. Any differences these 2 to 3 days make would be minimal. Weight changes from Days 21 to 28 were comparable for all groups. OK

### 211(b) Toxicology Research Group ETBE One-Generation Reproducti on Toxicity Study Report

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12 5/4/2010 

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Draft Report, Table 23 The Table number should appear on the Table itself and not just the cover page. No change possible due to Xybion limitations.

OK

Appendix A, Page 2 and 3 The duration of the temperatur e and humidity measurements that were out of range should be indicated. No temperature excursions. Incidence of humidity excursions noted. OK

Appendix A, Page 4, Frequency of Administration Typo (2 periods) in the middle of the paragraph. Corrected. OK

Appendix A, Page 7, Section 2.3.1: “Co n centration”. Corrected ok Appendix A, Page 20 The GSD value for April 17 is 1.663, I believe. Please verify. Corrected. OK

Appendix A, Page 26

The nominal value for March 20 is 2155 and should probably round to 2200. Please verify. No change.

Data indicates nominal value is 2150. ok

Appendix A, Page 35 The MMAD for May 22 should be 12.27. Please verify. Corrected. OK

Appendix A, Page 37 Please verify the nominal value for July 15. Nominal value reviewed and corrected. ok

Appendix A, Page 40 The temperature for May 16 sh ould be 26. Please verify. Corrected. OK

Appendix A, Page 31, July 3, Individual Analytical Chamber

Concentration The last value for this day is actually a mean of 2 values taken during a 7- minute span. Usually when a low or high value is noted, adjustments are made and additional assays are performed to verify that concentrations are indeed closer to target, and the original value is reported as it is the most representative for the time inte rval. Why was an average reported and why were all valu es not used? Please clarify. All values were reported as per laboratory SOP which states that when a sample is not within 20% of the target concentration, a repeat sample is taken. If the repeat sample is within 20% of the original sample, the original sample is reported. If the repeat sample is not within 20% of the original sample, a third sample is taken. All three samples are

evaluated and the 2 values closest in arithmetic agreement (providing the higher value is within 25% of the lower value) are averaged and this average is reported.

ok No adjustments are made to the system between the original and repeat samples. Appendix A, Page 47, Group 2The H-1 value is not reported. It was run after the second H3 measurement and was 2000 mg/m3. Corrected. ok

Appendix A, Page 49, Methodology The pathlength should be 3.39, I believe. The first setting was 7.00, but it was reset to 3.39 on 6/2/02. Please clarify. Corrected. OK

Appendix A, Page 49, Calibrations The calibration from 10/10/01 was used for this study as

well. Per the data, only 1 calibration (10/10/01) is included in the data and report. OK

Appendix A, Page 50, Calibration Checks

None of the absorbance values fell outside of the 15% range, so the rest of this paragraph is not needed. Deleted. OK

Draft Report, Appendix B, Page 25 The value reported for benzene in the control sample should be < LOQ and the value for Toluene should be ND to agree

with the raw data. Corrected. OK

Draft Report, Appendix B, Page 26 Corrected. OK

### 211(b) Toxicology Research Group ETBE One-Generation Reproducti on Toxicity Study Report

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13 5/4/2010 

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### Page
The value reported for the cont rol sample benzene should be

ND and the toluene value should be < LOQ to agree with the

raw data. Draft Report, Appendix B, Pages 27 through 30 Should state that these gas chromatograms were obtained from the Day 21 exposure samples. Indicated that the Gas Chromatograms were obtained from Exposure 21 Samples on Pages 27 through 30. OK Draft Report, Appendix B, Page 28 The retention time value for 3-Methyl-1-butane should be reported as 19.6 not 19.80. The retention times for trans-2- pentene, 2 methylpentane, 2,4 dimethylpentane and 2,3- dimethylpentane are not represented on the chromatograms. (These four compounds are also missing from the chromatograms on Pages 29 and 30.) No correction is made. Due to the limited space on the chromatograms, the retention times for trans-2-pentene, 2 methylpentane, 2,4 dimethylpentane and 2,3-dimethylpentane were not shown properly on the chromatograms. OK

Draft Report, Appendix B, Page 30 The retention times for N-pentane and 2,3 dimethylbutane do not agree with the raw data values (23.08 report vs. 23.19 data and 32.99 report vs. 33.11 data). Same as the above. OK

Draft Report, Appendix D, Page 15 Female #1555 is missing the Clinical Observation of Terminal Sacrifice (also Females #2559 and 2533; on Page 16 Females #4552 and #4562). Animal Nos. 1555, 2553, 4552 and 4562 were found mated and gestation physical observations were collected. Appendix D only includes non-mated females during the mating and post-mating periods.

Terminal Sacrifice for female No. 2559 added to Appendix D. OK Draft Report, Appendix D, Page 21

Females # 4553 observation st ates “accidental” should state

“accidental death”. Corrected (also Appendix C and Table 2).

OK

Draft Report, Appendix D and Table 2

The observation for Male Anim al #3055, “humane sacrifice, black/brown stains on snout and grasping” are reported in Appendix D as the 56 th day of the study, but Table 3 has these observations reported as Day 63. Also on Day 56 the observation of “within normal limits” is reported. This data should be reviewed and appropriate changes made to Table

2. Appears to be reported correctly. Animal was sacrificed on Day 59. When the death of an

animal occurs between scheduled observation intervals, the individual animal data (Appendix D) is presented at the previous interval (Day 56), but the summary table (Table 2) presents the data at the next interval (Day 63). OK

Within Normal Limits was noted for Animal No. 3055 during the scheduled observations on Day 56. Black/Brown Stains on Snout and Gasping were noted on Day 59 prior to sacrifice for humane reasons (Room Check obs). Notation added to preface page to Table 2; Preface page with explanatory notes added to Appendix D. OK Draft Report, Appendix D, Page 10 Male Animal #4055 “decreased food consumption and decreased fecal volume” are repo rted twice for this animal. The total days for this observation are not listed correctly in

Table 2. This data for all observations for this animal needs to be re-evaluated and tabulated correctly. Reported correctly in Appendix D and Table

2. Observations were noted at both the weekly physical examination and during the daily viability check (room check) and are categorized separately. The duplication in Appendix D indicates these observations were seen at both types of exam. Preface page added to appendix explaining this apparent duplication. OK

### 211(b) Toxicology Research Group ETBE One-Generation Reproducti on Toxicity Study Report

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14 5/4/2010 

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In Table 2, one set of these observations is summarized in the Miscellaneous category (physical examination), and the other in the Room Check category (viability check). A notation has been added to the preface page.

OK

Comment: Draft Report, Appendix G When an animal is no longer on study, a footnote should be added to clarify the missing weights – i

. e . Males #2073 and

3055. Footnotes added to Appendices G, H and M.

OK

Draft Report, Appendix P

The calculations for number of days in stage, number of cycles and mean number of days of cycle are calculated using the data obtained from Day 49 up to Day 69. The data from Days 70 to 83 has not been included in these calculations. If this data is included, the summary data will change ( i

. e . female #1559, the number of cycles changes from 1 to 4 and the mean number of days of cycle changes from 18.0 to 7.8). Appears to be reported correctly. Data is summarized for the 21 day pre-mating evaluation period only. Since the length of the co-habitation period varied from 1 to 14 days, including these data would skew the

results. Appendix P was regenerated to reflect the day on which estrus cycles were monitored within the exposure period. Estrus cycle monitoring started on test day 49 and continued for 21 days before mating was started. Preface indicates estrus cycling was evaluated for 21 days prior to mating and then up until successful mating. Only the 21 days prior to mating is used in the assessment. Agreed OK Draft Report, Appendix Q Should NA be added to the entry date of insemination, instead of leaving a blank space for the animals that were pregnant and a sperm/plug was not observed? It would seem that adding NA would imply the animal was not pregnant. Insemination DID occur; the laboratory staff just missed seeing any evidence (sperm/plug).

Ok Draft Report, Appendices R and S, Group 1 The values for Day 28 should be corrected to reflect the litters that were sacrificed early due to the rack overturning. These pups did not survive to Day 28. Also for Appendices

T and U the litters should be labeled that did not survive to Day 28. Appendix R – Litter Data. Notation added to Day 28. No other change necessary. ok

Appendix S – Sex and Status. Day of

sacrifice corrected for pups sac’d early. No other changes necessary. ok

Appendix T – Pup Clin Obs. Notation added. No other changes necessary. ok

Appendix U – Pup Body Wts. Notation added to Day 28. No other changes necessary. Ok Data, Draft Report, Appendix Y Some raw data printouts record the organ weights to three decimal places but Appendix Y reports the data to four places. Is the computer file the raw data or the computer printout? Per SOP, auto-print from the TASC data collection system is raw data. For this study, data collected on 16 and 17 Jul 02 was printed to 3 decimals and data collected thereafter (18 to 29 Jul 02) was coll ected to 4 decimals. Appendix Y was re-printed to 3 decimals to

match data from 16 and 17 Jul 02. ok Draft Report, Appendix Y Corrected. Ok

### 211(b) Toxicology Research Group ETBE One-Generation Reproducti on Toxicity Study Report

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15 5/4/2010 

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The final body weight used for the organ weight to body weight ratios was the Day 21 body weight not the Day 28 weight. These tables need to be recalculated using the Day 28 weight. Draft Report, Appendix Z Animal number 3055 is report ed as “elective sacrifice” when it was supposed to be change to “human sacrifice”. It was changed in the TASC system to “humane sacrifice”, but not in ZYBION. Please clarify. As well, shouldn’t a gross observation of “snout injury” have been recorded? Type of death corrected for Animal No. 3055. “Snout injury” was not recorded by the Necropsy Dept at the time of sacrifice,

therefore this cannot be added at this time. ok

Appendix Z, Animal number 4553

The fracture of the “head” was supposed to be changed to

“skull” but was not. Corrected. Ok

Appendix AA, Released Pup and Historical Control Data It would be helpful to indicate when the necropsies occurred for the 106 pups. It may also be helpful to indicate that dams

were in the inhalation chambers at the time of the accident. Lactation day of sacrifice for each litter is indicated on the preface.

Notation added that the dams were in the exposure chamber. Ok Appendix AA, Release Pup Data Is there any way of indicating on this printed out data that pups were at from day 25 to 28 of lactation instead of just indicating day 25? Also, as noted before, the historical data for pup body weights from study 4202 has used the Day 21 body weights, I believe. We cannot make this a range of days. ok

Terminal body weights for 4202 corrected to Day 28. Ok

Appendix DD: There were two Protocol Amendments, I

believe. The second should be added. Protocol amendment #2 has not been signed as of yet and will be resubmitted to API with the 2nd draft report. Ok...have now? Protocol Amendment 2: Please verify #3 / second paragraph Author references match those in report (Dunlap and Duffy 1975 is listed in amendment versus___ Armitage 1971 is in the report on p.30)

Amendment corrected to match the report

which has been modified for the ANCOVA analysis. Ok

DATA ISSUES

Data The animals for this study were housed in Room I-809 from June 17 through July 8 according to the data. The air change data is present for Rooms V- 708 and I-810. Air change data

for Room I-809 should also be available in the records for this study or a note should be in the data to explain the

situation. Please see protocol deviation dated 23 Jan

2004 which addresses this situation. ok

Analytical Data The GC Chromatogram printouts for Exposures 21 through 56 list the study number as 00-6129 instead of 00-4204. A study clarification note for the error was added to the analytical raw data. ok

Test Material receipt records Four, 50 cc cylinders were received on 11/6/01. These

records are included with this study data. Are we certain

these were filled with ETBE? Are more notations written on the original documents? Please verify that these are the correct receipt documents for this test material. The label for the contents of the archived cylinder is marked that it is G/ETBE. The disposition of the other 3 cylinders received that same day was also HLS archives for G/Baseline, G/ETOH and G/MTBE. ok

### 211(b) Toxicology Research Group ETBE One-Generation Reproducti on Toxicity Study Report

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16 5/4/2010 

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Test Material Usage Records It is indicated that large Cont ainer Number 1 was returned to sponsor, Chevron on 12/7/01 and Number 2 on 8/5/02.

Please correct this to indicate exactly where these tanks were sent. API is the sponsor. The location of Chevron should be given. As well, it is indicated that 1 cylinder was sent to the “archives” on 12/6/01. Which archives, HLS or EPL? Please clarify in the data. Test cylinder returns were made directly to Universal Environmental (trucking company hired by Chevron) and then they delivered to Chevron. We do not have a receipt showing exactly when and where the cylinders were then delivered but this was recently requested

from API and Chevron. The 1 cylinder that was sent to the “archives” on 12/6/01 was at HLS as per the data.

EPL archives received one cylinder retain from Chevron_same lot as used in study

ok

Test Material Usage Records An ending measurement of the small tanks needs to be

taken. The weights were transferred from the TMAS sheets to complete this data. ok

Parental Organ Weight Data The lung data for animal number 4563 was originally recorded as 15.5467g, then was changed to 0, then to **, then to 0 again on the computer. None of these changes were

recorded on the hand recorded edit sheet. The last change seen during the audit was to 0 and was done 1 month after the sacrifice. A value of 15.5460 g is reported, but it is kept from mean calculation. No explanation for this weight that is 10 times that of other animals is given in the report in Appendix X. Please add an explanation to the report, comment on the data trial, and make certain that data are trackable to that reported. The original weight recorded was a combined liver and lung weight and the edit to 0.0000 was intended to exclude the erroneous weight in the TASC system. It appears that the edit

from 0.000 to ****** was made in error and

was therefore corrected back to 0.0000. All organ weights were transcribed to the Xybion data collection system for reporting, where the lung weight for Animal 4563 was input as 15.5467. During editing to exclude this weight from calculations, the weight was inadvertently changed to 15.5460. On further review, it was determined that, since a valid liver weight was obtained, the lung weight could be calculated from the combined weight and reported. An additional edit was performed and an explanatory memo added to

the file. OK Where did separate liver weight

come from? The reported liver weight was recorded directly into the TASC system at necropsy

OK Apparently, Animal number 20 70 was “sacrificed” on the computer system too early. Th e animal was “resurrected” and data was re-entered according to the computer data correction sheets. It then appear s that data for number 2070 was removed because the wrong animal number was selected when data was added. The data may have belonged to animal 3074? A necropsy sheet was found for number 2070, but no print-out was found with 2070’s observations that had been deleted. As well, no findings for number 3074 were found. Please verify that the data trail is complete for these

errors and corrections and that it is clear to a reviewer what happened. This is incorrect. According to the Computer Raw Data Change and Addition form, necropsy observation data for Animal No.

3074 was entered as Animal No. 2070. In order to remove this data, Animal No. 2070 was “resurrected”. This single edit removed all necropsy observations as well as the terminal body weight. The next entry on the

CRDCA form was to re-enter the terminal body weight for Animal No. 2070.

Necropsy data for Animal No. 2070 was collected manually (on a Necropsy Record Sheet) and entered into the Xybion system

once the animal was “resurrected”.

### 211(b) Toxicology Research Group ETBE One-Generation Reproducti on Toxicity Study Report

Reviewer Checklist

17 5/4/2010 

### Comments Action/Response 

### Page

Data for Animal No. 3074 was captured directly into the Xybion system (albeit as Animal No. 2070) therefore no NecropsyRecord Sheet exists for this animal. A printout dated 10 Jul 02 shows data for Animal No. 2070 (actually No. 3074) collected on and entered on 9 Jul 02. The data was input under

the correct animal number when 2070 was

edited.

Print-outs showing the corrected necropsy

observation data for Animal Nos. 2070 and 3074 and the audit trail “resurrecting” Animal

No. 2070 have been added to the data . OK

Data The printout for the weight of food left is missing for June 20, 2002. Food left weights for this date are located in

data records Volume II Book 5. ok