### 211(b) Toxicology Research Group MTBE Two-Generation Reproducti on Toxicity Study Report

Reviewer Checklist

1 5/6/2010 

### Comments Action/Response Page

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. However, two issues raised by the peer review and the API response should be revisited.

This study has concluded a reproductive NOEL of 20,000 mg/m 3 for the Gasoline MTBE Vapor Condensate. Statistically significant incr eases in epididymal sperm concentration and percent abnormal sperm were observed at 20,000 mg/m 3 exposures, but were dismissed based on historical control data. In his review, Dr. Schlesinger noted that concurrent controls should have been used, although the Study Group declined to adjust their results accordingly. We agree that the use of histor ical controls to supplant statistically significant findings is inappropriate and would respectfully submit that the reproductive NOEL be revised downward to 10,000 mg/m 3

. This issue could be resolved by designating 20,000mg/m 3 as the NOA EL See comments to Dr. Schlesinger below.

Summary and R/D revised as suggested

. OK_

Page 25. As noted by Dr. Goldsworthy further discussion of the wide variation in environmental parameters of temperature and especially humidity is warranted. The potential adverse effects of ultra low humidity (1.88% R.H.) on an inhalation study are of concern. Were the animals exposed to such conditions for significant lengths of time? Appropriate additions to the text will be made by the study director.

Added discussion of excursions added to the

text in 2.9.9. OK

Page 48: "developmental" is misspelled "devolopmental". Corrected Ok



### Peer Reviewer – Schlesinger

General Comment The exposure group numbering is not consistent in the report. In some places this is given as I, II, III, IV and in others as 1, 2, 3, 4. This inconsistency will be brought to the attention of the study director and, where practical, adjustments can be 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. Revised for consistency where practical. Ok

Page 37. 2.14.1. It is noted that if significant differences were found among

the means then additional "tes ts" were performed. In actuality, there appears to be only one such test. In addition, why were results at each time point analyzed separately rather than using a two way analysis of variance that would

include time interval as a factor? Only 1 test (the Dunnetts t test) was performed to compare the group means from the control mean. The report will be thus

clarified. Ok

### 211(b) Toxicology Research Group MTBE Two-Generation Reproducti on Toxicity Study Report

Reviewer Checklist

2 5/6/2010 

### Comments Action/Response Page
Data was statistically evaluated per protocol per the standard statistical package included with the TASC data capture software. Report

OK as is.

Why were two levels of significance used on some tests and not on others. The level should be determined prior to the start of the study. Only 1 test (the Dunnetts t test) was conducted at both the 5% and 1% significance levels. Although not stated in the protocol, these levels of significance were preselected since they are inherent in the laboratory's computer statistical package. Comparison at 2 levels of significance has been common practice for this laboratory. Ok

It is noted that statistical evaluations were not performed when the standard deviation for the control group was 0. However, this resulted in the inability to indicate statistical significance in some of the resu lts. There are other tests that could have been performed that would have allowed statistical comparison of test animals to controls even when

there was no variation in the control data. The standard deviation for an endpoint would be 0 if the n=1, or all of the individual values

were the same. This is a standard statement

for the statistical methods section. These events occur infrequently, usually in macro/micro pathology observations, and it was not necessary to consider alternative

statistics. Ok

Page 37. 2.14.2. The last paragraph of this section seems out of place here. [in a discussion of statistics of sperm and ovary data] The last sentence to which Dr. Schlesinger refers, deals with macro/microscopy of the testes. The RG agrees that this sentence would be better placed elsewhere, specifically in section 2.13.5 Sperm counts, motility & morphology.

Deleted as already in section 2.13.5. OK

Page 40. 2.17. There appear to be an excessive number of technician-related errors in this study. Some explanation is needed. Nearly all of the protocol deviations were

related to isolated errors in data collection. None significantly impacted the overall results or conclusions of the study. While the absolute number of protocol deviations may seem excessive, a Two-Generation Reproduction study is a considerable undertaking and involves a very large number of animals. The fact that these protocol

deviations were noted and documented reflects positively on the Quality Assurance practices that have been put in place by API, and at the laboratory. No netheless, the RG has brought this issue to the attention of the laboratory and will strive to minimize future protocol deviations. Ok

Page 42. 3.1. The explanation given for the difference between the

measured and nominal concen trations involves assumed 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

### 211(b) Toxicology Research Group MTBE Two-Generation Reproducti on Toxicity Study Report

Reviewer Checklist

3 5/6/2010 

### Comments Action/Response Page

inaccuracies in the calibration of chamber airflow and the IR monitor. There is no reason why the IR monitor in particular should not have been accurately calibrated using accepted procedure. A similar comment applies to the airflow

measures. have been due 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.

This discussion has been simplified to delete speculation about any errors.

What accounts for the particles in the chambers, especially

the control chamber.

Was a filter used for incoming air? 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 43 indicates that the particle concentrations in all of the chambers ranged from about 3 to 4 micrograms 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

Page 45. 3.2.4.

The differences in feed consum ption appear to be just as evident in females as in males, although the comment in the

text states otherwise. The report states that exposure-related decreases in feed consumption during the initial 7 weeks premating occurred in F1

animals of both sexes but was more evident in males. In comparing these data, although absolute consumption in treated animals of both sexes was similar, the female controls consumed more than male controls, reducing the statistically signif icant differences with treated groups. While 10000 and 20000mg/m 3 F1 male feed consumption was significantly decreased during all 7 weeks, statistically significant decreases was seen in females exposed to 20000mg/m 3 only at wks 1, 3 but more frequently at 10000mg/m 3 in wk 1, 3-6. . Given the variability of these data, the RG accepts the study director’s evaluation Ok

### 211(b) Toxicology Research Group MTBE Two-Generation Reproducti on Toxicity Study Report

Reviewer Checklist

4 5/6/2010 

### Comments Action/Response Page
of this parameter.

Page 46. 3.2.7 and Table 9. From the table, there appears to be a decrease in maternal lactation body weight at the high exposure level on days 21 and 28. This does not seem to be mentioned in the body of

the text. The RG will request the study director to report these data and address the significance

in the text.

Discussion added re this difference. OK_

Page 48. 3.4.1 and Table 20. The pulmonary effect noted, na mely discolored foci, appears to be similar in both genders. As noted, there was a large increase in lesion incidence at the 10,000 and 20,000 levels. However, due to the fact that there were no such lesions in control, no statistical tests could be performed. This relates

back to the earlier comment ab out using another statistical

test. Control (P0 and F1) and 20000mg/m 3 P0

males had 0 incidence of discolored foci. The RG accepts the discussion of the discolored foci in treated animals, based on observed incidence, as sufficient to address the occurrence of these lesions without employing statistical analysis. Histological identification of alveolar macrophage aggregates in P0 and

F1 rats correlated with occurrence of discolored foci. The increased frequency of this background lesion was plausibly related to test substance exposure but was considered of minor clinical significance by the attending
pathologist



### . OK_

Page 49. 3.4.2 and Table 22. The investigators concluded that the decrease in lung weight

noted in the table did not occur in a "treatment-related pattern." However, to this reviewer, it did appear to follow such a pattern, in that all exposure levels were above control. Furthermore, if as noted, the microscopic findings would have expected to result in in creased lung weight, perhaps some explanation should be given as to why this effect was

not observed. As indicated by Dr. Schlesinger, the absolute lung wt in F1 males did decrease in a statistically significant treatment-related manner. However, when lung wts were compared relative to final body wt, only the 10,000mg/m 3 relative wt was statistically significantly lower compared to controls, indicating that the organ wt changes related to body wt did not occur in a treatment –related manner. Furthermore decreased lung wt were

not seen in P0 animals of either sex or in F1

females. The incidence of alveolar macrophage aggregates seen in lungs of F1 males was generally lower than for other groups. There does not seem to be a substantive linkage be tween decreased lung weights in F1 males and discolored foci or alveolar microphage aggregates, leaving the clinical significance of these lower weights

indefinable.

Additional discussion added. ok

Page 50. 3.4.3. The investigators note that th e increase in the number of sperm/g of epididymis and the increase in the percentage of abnormal sperm in the Po generation were not considered

treatment related since the m ean value of abnormal sperm was within the range of histori cal controls even though the increase at the high exposure level was statistically The biological significance of increased sperm/g epididymis and increased % abnormal sperm can be better evaluated when actual increases are presen ted in report text. Although the increased sperm count in P0 males is 15% higher than controls (891.2 vs. 753.6 million sperm/g epididymis), this value OK_

### 211(b) Toxicology Research Group MTBE Two-Generation Reproducti on Toxicity Study Report

Reviewer Checklist

5 5/6/2010 

### Comments Action/Response Page
significantly different from control in this study. This reviewer finds this type of reasoning to be faulty. If an effect is noted using the controls for the specific study, then what is the justification for conclu ding that the effect is of no biological significance merely because of the historical control data. This leads to the obvious question of what is the purpose of the controls in th e study if when an effect is found, it is ignored if past controls show no such effect. Thus, this reviewer considers the effect on sperm in this study to be biologically significant. is similar to control rats in F1 generation (887.5) and the historical control average at Pathology Associates Inc (PAI – 869.9). The statistical significance is produced by the low value for control sperm in the P0 generation as is demonstrated in the study itself and not by

historical control data alone. The increased % abnormal sperm at 20000mg/m 3 P0 rats is only 0.7% greater than controls (2% vs. 1.3%) and although statistically significant, falls

within the historical ra nge at PAI (0-6.7% in control animals). In cases of statistically significant small changes at single doses or changes which appear statistically significant because control values are unusually high or low, it is appropriate to consider these events in relation

to historical data to determine biological/clinical signifi cance. Only a small sub-set of a population of animals is used in an individual study, and where changes are minimal, that data must be evaluated in a broader context. This approach is similar to evaluating results from medical tests in

humans where the individual’s values are considered in relation to the population range for that endpoint as well as the patient’s

baseline value if available from earlier tests. Interestingly, Dr. Goldworthy also addressed these data and suggested that the historical control values be included in the text (see comments below). The RG accepts the study director’s evaluation of these male reproductive endpoints but will direct that the control data be included in the report text as well as in Appendix CC.

HCD added to report text section 3.4.3.

GFAP Study Page 17. In this study, only one level of significance is used, compared to the main study as noted above. Thus, there appears to be some internal inconsistency. The GFAP study was performed by Dr. O’Callaghan, a different investigator, from the main study and selection of statistical methods conformed to those used in his laboratory. The RG has requested Dr. O’Callahan to clarify the statistical choices.

Statistical discussion clarified by JOC. OK_

Why did the investigators not perform a two way ANOVA See above OK_

### 211(b) Toxicology Research Group MTBE Two-Generation Reproducti on Toxicity Study Report

Reviewer Checklist

6 5/6/2010 

### Comments Action/Response Page
that included gender as a factor instead of a one way? Statistical discussion clarified by JOC.

Tables 5 and 6. Perhaps the * notation for footnote should not be used since it is easily confused with notation for significant difference. Symbol revised to #. OK_



### Peer Reviewer – Goldsworthy
The study, including exposure methodology, was properly conducted and adequate to test the potential reproductive toxicity of Gasoline MTBE. The study deviations identified would not affect the overall study integrity or conclusions. The temperature and humidity values demonstrated significant variation (up to 31.3 ° C in temperature and 2-98% in humidity) and warrant additional comment in text to help explain these signification variations. Added discussion of excursions added to the

text in 2.9.9. OK_

Exposures were appropriate, consistent, and reasonably close to the targeted exposures. No gasoline MTBE exposure related effects were seen in the male or female reproductive systems, including mating behavior, conception, gestations, parturition, lactation and weaning, growth and development of offspring, estrous cycle, and gonadal functions. Similarly, no exposure-induced changes were noted in follicle counts, sperm counts, motility or morphology, neonatal morbidity and mortality, and prenatal and postnatal developmental toxicity. Similarly, no treat ment effects were noted in

survival. GFAP levels were al so examined in specific brain regions. Exposures to gasoline MTBE did not elevate GFAP levels in either males or females of the first generation exposed rat pups (editorial comments made previously in GFAP report remain). No comment necessary

Clarification of statistical methods as requested for previous reports will be incorporated. In earlier comments about the subchronic study on baseline gasoline, EPA agreed that there were no major technical problems with the GFAP section of the report (including format), but that a request for

additional statistical information was

reasonable.

This was added to page 17 of the GFAP

report. OK_

There were no exposure-related decreases in feed consumption values in F1 animals during the premating period. No exposure-related differences were seen in macroscopic evaluations except for the increased incidence of pulmonary foci of discoloration in treated animals compared to exposed rats. Organ weight differences were observed in the kidney of male and female rats; microscopicfinds in the male rats were consistent with hydrocarboninduced nephropathy and generally not considered to be relevant to human risk assessment. Test was vague in presenting or discussing any corresponding histological findings to explain the increas ed renal weights in treated female rats; text should clearly state if or if not microscopic changes were noted in female rats. Increased liver weights were observed in high-dosed male and female rats in F1 male rats; microscopic findings were not found. Both sexes exposed to gasoline MTBE exhibited an increase in the First, in Table 22, female absolute kidney wts were not statistically significantly higher than controls for P0 or F1 parental animals. Only the organ/body wt ratio for F1 parental females exposed to 20000mg/m 3 was significantly increased at p< 0.05. Secondly, Tables on p. 51 (P0 rats) and p. 52 (F1 parental rats) show that no histological findings were observed in female kidneys of either generation at 20000mg/m 3 level of

exposure. No adjustments to the text are considered OK_

### 211(b) Toxicology Research Group MTBE Two-Generation Reproducti on Toxicity Study Report

Reviewer Checklist

7 5/6/2010 

### Comments Action/Response Page
incidence of aggregates of intra-alveolar macrophages. necessary.

In summary, experimental procedures and exposures were adequate to test the potential reproductive toxicity of gasoline MTBE level in rats. Based on the above noted findings, a NOEL [for systemic toxicity] was not established. No effect at any exposure a ffected reproductive performance of parameters, including mating, fertility, gestation, parturition, lactation and weaning, growth and development of offspring, estrous cycle, follicle counts, or gonad functions. Thus, with respect to reproduction performance, a NOEL of 20,000 mg/m 3 of gasoline MTBE was concluded. I concur with the report conclusions. No comment necessar 

### y Ok

NOAEL of 20,000 mg/m3

Editorial Suggestions Summary, page 8 and Results, page 49 (section 3.4.2) state that differences in kidney weights from male and female rats "were consistent with the microscopic findings discussed in the following section." This is true only for the male but not the female induced changes. First, in Table 22, female absolute kidney wts were not statistically significantly higher than controls for P0 or F1 parental animals. Only the organ/body wt ratio for F1 parental females exposed to 20000mg/m 3 was significantly increased at p< 0.05. Secondly, Tables on p. 51 (P0 rats) and p. 52 (F1 parental rats) show that no histological findings were observed in female kidneys of either generation at 20000mg/m 3 level of

exposure. No adjustments to the text are considered

necessary. OK

Section 3.4.3, pages 49-50. Text should note the laboratory 's historical control mean values and range so that comparisons can be made to the significantly increased values in the number of sperm/gram and percentage of abnormal sperm from exposed rats. Historical control values from PAI are supplied in Appendix CC. The RG can instruct the study dir ector to include these values in the report text.

HCD added to report text section 3.4.3. OK_

Page 27, section 2.13.7. Test states that "sponsor was responsible for the spirit of GLP compliance' of the subcontractor. It is not clear "spirit of GLP" is defined and how it is assessed? The inspections were noted on page 4-5 of the GFAP report. The spirit of GLP is defined as collection and maintenance of accurate data as is expected of any careful investigat or with appropriate review and documentation. Inspections by the RG quality assurance expert have been performed and dates of inspections and data review will be provided in the final report. OK_

Page 54, Conclusions. Text appears to be missing a sentence immediately prior to the last sentence in the conclusions. The missing sentence must state the summary data (i.e. not effect at any exposure on reproductive performance in study...) before launching

into the last sentence with a "Therefore". Missing sentence added and clarified and same for Summary section. OK_

### 211(b) Toxicology Research Group MTBE Two-Generation Reproducti on Toxicity Study Report

Reviewer Checklist

8 5/6/2010 

### Comments Action/Response Page 211(b) Research Group Reviewer

General Comment

Unlike previous reviews, this review incorporated only the text, summary tables, and Appendices P and CC, and did not include a QA-type of evaluation of the data. Hopefully, those needs were covered by other reviewers. This has been particularly important on several studies for the counts of

total stillborns. Noted OK_

There are gross observations of pulmonary foci and microscopic observations of intraalveolar macrophages. Since these are attributed to exposure, these should have a line of explanation regarding th eir interpretation in relation to toxicity. These observations are discussed in both the Summary and the R&D in relation to the systemic NOEL discussion. ok

The study does not have an overall NOEL, but does have a reproductive NOEL identified. API/211b work group should

determine if we want to have a NOAEL identified. Report Okay as written_do not need to identify systemic NOAEL ok

Editorial Suggestions Page 6, Summary, last line: Please revise “reasonably” to “acceptably,” since the variation of measured concentrations from target concentrations has an acceptable range. Revised to ‘acceptably’ although there was not an established acceptable range. OK_ (p.6 4 th

line )

Page 7, 1 st – 5 th lines: Please delete the two sentences that address possible reasons for differences between measured and nominal concentrations. Since the data were acceptable (and usually very good), these sentences provide unnecessary information. Deleted. OK_

Page 8, lines 15 – 18: Please add a sentence to describe the meaning of pulmonary foci.

The same applies to the microscopic observation, increased

incidence of intraalveolar macr ophage aggregates (lines 29 – 31). These are standard pathology terms in conventional use and are self-defining. OK_

Page 20, section 2.3.7, Analysis: I feel uncomfortable with the 211b studies lacking test material characterization information. If this isn’t added, there should at least be

identification of the reference for the test material characterization report. Added reference to EMBSI report. OK_ (p. 18 2.4.7)

Page 21, section 2.4.9, Dispensing: I believe at the time of test material shipment, we we re known as Chevron Research and Technology Company. Company name corrected. 21 OK_

### 211(b) Toxicology Research Group MTBE Two-Generation Reproducti on Toxicity Study Report

Reviewer Checklist

9 5/6/2010 

### Comments Action/Response Page

Page 23, section 2.9.2, Housing: Please correct typographical error: “...housed during the during initial

week...” Corrected. 24 OK_

Page 29, section 2.12.6, Physical Maturation Landmarks: Please revise to: “and the body weight of each pup was

recorded when the criterium was met.” Corrected. 30

“criterion”

used

Page 40, section 2.17, Protocol Deviations, No. 12: Please indicate how measurements would have been altered by being collected post-fixation rather than immediately postperfusion. I have compared these results vs results from study 00-4207 for differences and there were none; therefore, this error seems to have made

no difference in the results. No change in the report

written in

explanation

in checklist

Page 42, section 3.1, paragraph following table: Please revise “reasonably” to “acceptably ,” since the data needed to meet acceptance criteria/um. Pl ease delete the second and third sentences in this paragraph as unnecessary. All done. OK_

Page 43, section 3.1, 5 th line after the particle size table: Please revise “a reasonably” to “an acceptably.” Done. OK_

Page 44, section 3.2.2, Physical Observation Data: Three rats in the 20K group had a red vaginal exudate (none in any other group). Since this is a reproduction study, please note what these findings were and/or what they were related to (i.e., if observed shortly before/after parturition). Additional discussion added.

Page 44, section 3.2.2, Physical Observation Data: Three rats in the 20K group had a red vaginal exudate (none in any other group). Since this is a reproduction study, please note what these findings were and/or what they were related to (i.e., if observed shortly before/after parturition). Same as above. ok

Page 45, section 3.2.4, Feed Consumption: The feed consumption data were not dose-responsive (Table 11); please comment. Clarified that ’not in an exposure-level-related pattern’. OK_

Page 46, section 3.2.5, Estrous Cycle Data: Pseudopregnancy cycles appear to be included in the calculation of mean group va lues and the intergroup statistical comparison. A later reproduction study had a very nice write-up of estrous cyclic ity that eliminated these artifactual data from analysis. Please revise the presentation The tabulation has been revised to be consistent with other reports. ok

### 211(b) Toxicology Research Group MTBE Two-Generation Reproducti on Toxicity Study Report

Reviewer Checklist

10 5/6/2010 

### Comments Action/Response Page
and analysis in a similar manner for this study. Although pseudopregnant animals occurr ed in all groups, they contributed to the large standard deviations that reduce statistical power.

Page 48, section 3.4.1, Macroscopic Postmortem Evaluations: The table for Discolored Lung Foci does not add up to Table 20, page 4 (20K males had 1, not 2 discolored foci in the F1 generation) and page 5 (10K females had 5, not 6, while 20K females had 15, not 17, in the F1 generation). The numbers DO add up if the observation “Discolored Lungs” are added to the Discolored Lung Foci values, but this is not how the table is presented on page 48. Discolored foci was removed from the table title and replaced with discoloration. A

footnote was also added to the table that states it includes discoloration, discolored areas and

discolored foci. Table 20 matches Appendix W. ok

Page 49, section 3.4.2, Organ Weights, 3 rd paragraph: Decreased lung weights did occur in a dose-responsive pattern, although the position that the microscopic effects associated with exposure , increased incidence of macrophage aggregates, would have increased organ weight, can be accepted. Please consider revising the text to be a bit

clearer. Clarified the discussion. ok

Page 50, section 3.4.3, Sperm Evaluations: Although the values for abnormal sperm can be found in Appendix CC, please consider adding them as shown, as well as a sentence or phrase that states that no increase in morphologically abnormal sperm was observed in the F1 males, as shown (additions underlined): “The mean value of abnormal sperm, 2%, is within the range of abno rmal sperm reported by PAI’s

Historical C ontrol (0 – 6.7%). As a result, the increase in the number of sperm/gram of epidid ymis and the increase in the percentage of abnormal sperm in the P0 generation, which was not observed amongst F1 males, were not considered

treatment-related. Added the HCD to the text and added

additional discussion. ok

Page 51, section 3.4.6, Lungs: Please add a clarifying

sentence to indicate the toxico logical significance of the tan pulmonary foci/aggregates of intraalveolar macrophages. Clarified. OK_ (p50)

Page 52, section 3.4.6, Alveolar Macrophage Table: The second row is missing its descriptor. Added. OK_

Page 53, section 3.4.6, Lungs: Same comment as above for p. 51. Clarified. OK_

### 211(b) Toxicology Research Group MTBE Two-Generation Reproducti on Toxicity Study Report

Reviewer Checklist

11 5/6/2010 

### Comments Action/Response Page

Page 53,section 3.4.6, second Alveolar Macrophage Table: The second row is missing its descriptor. Added. OK_

Page 54, section 4, Conclusion: Same comment as captured under General Comments: Does API want a systemic

NOAEL identified? Report Ok as written_do not

need to identify systemic

NOAEL

Table 5, page 5: The header reads “P0” but should read

“F1.” Generation corrected in Table 5 header OK_

Table 6: Please include a ro w for cumulative overally body weight gain. Cumulative body weight gains (Weeks 0-16 and Days 35 to 154) added to Table 6 and Appendix H (males only) OK_ Append H 0n

P5 which is 39/511 pps

GFAP Report: My only comment is that the Quality Assurance Statements on pages 4 & 5, are blank and should have information presented. Requested Chris Sexsmith to provide for

GFAP Lab and received 23Feb09 and added to report. We will provide for our lab? ok



### 211(b) Research Group QA/QC Reviewer
A. Draft Final Report and Associated Raw Data 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 has not been comp leted, it must be included in the sponsor’s compliance statement. Statement revised. 2

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 pr epared for the GFAP report.

These items must be included in the statement. Statement revised. OK 2

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 to the QA Statement. OK

Page 19, Section 2.3, Experimental Outline. The table, as presented in Section 2.3, indicates that 10 animals/sex/group from the P0 and F1 generation low and middle dose parental groups had reproductive tissues evaluated microscopically. Appears to be correct as is. Footnote’b’ indicates reproductive tissues were examined for Control and High exposure P0 and F1 parental animals and lungs from all groups 19

### 211(b) Toxicology Research Group MTBE Two-Generation Reproducti on Toxicity Study Report

Reviewer Checklist

12 5/6/2010 

### Comments Action/Response Page
Microscopic evaluation in these two groups was limited to the lungs (all parental animals except non-pregnant females) and selected gross lesions. were examined. OK

Also, for the P0 and F1 parental groups, additional protocolspecified tissues (adrenals an d pituitary) were examined microscopically from the ran domly selected 10 animals/ sex/group from the control and high-exposure groups. The experimental outline does not indicate that these tissues were

evaluated. Added to footnote ‘b’. OK 19

Page 22, Section 2.5.7, Weight at Initiation of Exposure. The weight ranges for the P0 generation males (182 to 247 grams) and females (126 to 188 grams) were not strictly within the protocol specified ranges of 150 to 200 grams for males and 125 to 175 grams for females. These departures from protocol specifications should be addressed in the data and report. Notation added that animals outside of the protocol nominal ranges were used at the discretion of the Study Director ‘as per protocol’. OK 22

Pages 40 and 41, Section 2.17, Protocol Deviations. The following typographical errors and omissions were noted in the protocol deviation section of the report:

a. Item 1: The animals transferred to the incorrect cage following exposure on June 18, 2001 were P0 (not F1) generation animals. Also, animal no. 2059 should also be included in the listing of animal numbers for

this deviation.

b. Item 2: The animals not exposed on August 24, 2001 were from the P0 (not F1) generation.

c. Item 4: Animal no. 2556, for which kidney and left ovary were lost at necropsy, was from the P0 (not F1) generation. Also, this deviation should state that no weights were collected for these organs.

d. Item 5: The referenced mating pair (animal nos. 3012 and 3507) was from the F1 (not P0) generation. Also for the male, the affected food consumption interval

was Week 13.

e. Item 6: The deviation states that adrenals were lost at necropsy for animals 1005 and 1007, and therefore not available for weighing and tissue collection. The data

indicates that this is a true statement for animal 1005, but for 1007, there is indication in the gross pathology data (see Appendix W) that the missing adrenal was due to agenesis (a congenital or developmental finding and therefore not a deviation?), rather than being lost at necropsy. The data should be reviewed and the deviation section revised as needed to reflect the study

records. Also, no written documentation of the deviation for

animal 1005 was found in the data at the time of this a) Corrected OK

b) Corrected OK

c) Corrected OK

d) Corrected. OK

e) Animal No. 1007 removed from deviation. Memo dated 15February02 documenting this

deviation is located in the data. OK 40-41

### 211(b) Toxicology Research Group MTBE Two-Generation Reproducti on Toxicity Study Report

Reviewer Checklist

13 5/6/2010 

### Comments Action/Response Page

audit.

f. Item 8: the referenced animal number (2023) was from the F1 (not P0) generation.

g. Item 9: The referenced animal number (4522 was from the F1 (not P0) generation.

h. Item 10: At the time of this audit, no written documentation of this deviation (processing and examination of brain, liver , spleen and kidney) was found in the study records.

i. Item 13: The referenced animal number (1520-03 was from the F2 (not F1) generation. f) Corrected OK

g) Corrected OK

h) Memo dated 20 May 02 documenting this

deviation is located in the data. OK

i) Corrected to 1510-03 in report as per protocol deviation memo. OK

Page 44, Section 3.2.2, Physical Observation Data, Line 10: “keratoacanthoma” is mistyped. Corrected. OK

Page 46, Section 3.2.7, Maternal Body Weights – Gestation

and Lactation Periods.

In line 3, the text should indicat e that F1 parental females in the 20000 mg/m 3 group were statistically significantly lighter at Gestation Day 0. Corrected. OK

Page 47, Section 3.2.9, Parturition and Litter Data. In line 6, the text references a 21-day survival (lactation index). Shouldn’t this be 28-day survival, since weaning occurred on day 28 of lactation? No – as per the calculations of the TASC system (listed on page 56), Day 21 is the proper interval for this index. OK

Pages 51 and 53, Section 3.4.6, Histopathological Evaluations. In the text tables that summarize the histologic findings in kidneys of P0 and F1 parental rats, the number examined appears to include on ly the 10 control and high dose animals randomly selected for microscopic evaluation. As reflected in Table 24, some groups also had animals for which the kidneys were examin ed due to the presence of gross lesions and the same diagnoses made. The text tables

should either be revised to include these additional animals

or clarification added which states that the summarization excludes animals for which kidneys were examined due to the presence of gross lesions. The text table revised to include clarification

which states that the summarization includes only the 10 control and high dose animals randomly selected for mi croscopic evaluation.

OK

Table 1, F1 Generation, Summary of Survival and Pregnancy. For group IV, the table indicates that no males died during the post-mating period. The study data indicates that animal 4002 was found dead on 1/25/02. It appears that

this animal should be includ ed as a post- mating death.

Please correct or clar ify as appropriate. Table revised. OK Table 1, page 2

Table 2, F1 Generation, Summary of Clinical Observations, Reviewed and appears to be correctly Table 2,

### 211(b) Toxicology Research Group MTBE Two-Generation Reproducti on Toxicity Study Report

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14 5/6/2010 

### Comments Action/Response Page
page 6. For the males, the total numbers of animals terminally sacrificed are report ed as 18, 15, 16 and 16 for groups 1, II, III and IV, respectively. These numbers do not

account for all male terminal sacr ifices. As per Appendix C, the total numbers terminally sacrificed in the male groups were 25, 26, 26 and 24, respectively. reported. Last scheduled weekly clinical observation was on Day 154. Additional

terminal sacrifices that occurred after this interval (Days 155 and 156) are therefore not

on this table. OK, because of the limit of the

table page 6

Table 5, Mean Body Weight Values, Page 5. The table header for body weights starting at day 35 should indicate “F1 Generation” (not P0). Corrected. OK Table 5, pages 5-6

Table 14, Summary of Delivery and Litter Data, P0 and FI Generations, pages 2 and 6. The table reports a 21-day survival (lactation) index for P0 and F1 pups. Since weaning occurred on day 28, the survival index should reflect a 28- day lactation period. The tables should be revised to include

this survival interval. No change possible due to system limitations.

OK Table 14, pages 2 & 6

Table 17, Summary of Pup Clinical Observations During Lactation, F1 Generation, page 1. According to Appendix T, a clinical observation of ulceration was noted for pup 2567- 10 on day 0 of lactation. This observation does not appear to be reflected in Table 17. Please correct or clarify as appropriate. Also, the table title on pages 1 and 2 of Table

17 indicate that incidences are presented as “frequency/ animals”. It appears that data are presented as “frequency/no. of affected litters”. Table revised to include Day 0 observations.

OK

No change to table title possible due to TASC system limitations. OK Table 17, pages 1-2

Table 23, Summary of Statistics for Absolute Organ Weights, F1 and F2 neuropath animals, pages 1 and 2. Footnotes for statistical signifi cance notations appear at the bottom of the page, although it does not appear that any significant differences occurred. Should the footnotes be

deleted? These footnotes are applied by the Xybion computer system and cannot be suppressed.

OK Table 23, page 1-2

Appendix A, Inhalation Report. The table numbering for this appendix is unclear. There is a Table I, II, III and so on, but there is also a Table 1 (Chamber monitoring Results). As well, the preface for the Chamber Monitoring Results indicates that it is Table II, while on the table itself it is indicated Table 1. Please clarify. Appendix reviewed and erroneous table numbering removed from Chamber Monitoring Results tables. OK Appendix A, pages 27-78

Appendix A, Inhalation Report. In the text of this appendix, I could not find a reference to the chamber monitoring results. Am I missing a page, or is this needed? Reference added. OK Appendix A, page 2

### 211(b) Toxicology Research Group MTBE Two-Generation Reproducti on Toxicity Study Report

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15 5/6/2010 

### Comments Action/Response Page

Appendix A, Inhalation Report, page 1, Temperature and Humidity. It would be helpful to indicate the frequency and

duration of the excursions from desired values. Frequency of excursions added. OK Appendix A, pages 2-3

Appendix A, Inhalation Report, page 3, Frequency, paragraph three. When F1 males and females were started on treatment at weaning, what day was that? And, was it the same day for each animal? Please clarify. Treatment initiated for each selected F1 animal on the day of weaning (Day 28). Section revised to add day number. OK Appendix A, page 4

Appendix A, Inhalation Report, Table V. The calibration of the Miran done in January of 2001 was used even though exposures were not initiated until June of 2001. This should probably be reported. Notation added to report. OK Appendix A, page 84

Appendix A, Inhalation Report, Table 1, page 7. Humidity value for Oct. 27 should be 39, please verify. Corrected. OK Appendix A, page 33

Appendix A, Inhalation Report, Table 1, Page 15. The second value for July 5th should be 1180. That was the 1.5 hour value. It appears that the system was adjusted and 2

minutes later the value was 1670. 1180 is a better estimation of the concentration for the previous 1.5 hours. Data reviewed. Since the sample taken at 0937 (1180 mg/m3) was not within 20% of the target concentration, a repeat sample to

confirm the result was taken at 0939 (1670 mg/m3). Since this sample was not within 20% of the original sample, a second repeat sample was taken at 0944 (1440 mg/m3). The repeat samples were averaged and the average reported (report corrected to 1560 mg/m3). System adjustments occurred at 0818 and 1020, not between the original sample and the repeats. OK Appendix A, page 41

Appendix A, Inhalation Report, Table 1, Page 15. As noted in previous item, for July 6th, the first value should be 1320 as the system had been running for a half hour. The 1640 value was 4 minutes after an adjustment was made. Same as previous response. Original sample taken at 0758 (1320 mg/m3) was repeated at 0802 (1640 mg/m3) and repeated at 0806 (1670 mg/m3). Repeat samples were averaged and reported (report corrected to 1660 mg/m3). System ad justment occurred at 0822, not between original sample and

repeats. OK Appendix A, page 41

Appendix A, Inhalation Report, Table 1, Page 19. The third value for September 30th should be 2310. This value was corrected on the CMR 2 days af ter exposure, but values had already been entered in the computer. No one verified changes during QC. Corrected. OK Appendix A, page 45

Appendix A, Inhalation Report, Table 1, Page 20. The fourth Oct. 23rd value should be 1990. Please verify. Corrected OK Appendix A, page 46

### 211(b) Toxicology Research Group MTBE Two-Generation Reproducti on Toxicity Study Report

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16 5/6/2010 

### Comments Action/Response Page

Appendix A, Inhalation Report, Table 1, Page 27. The

nominal value for June 23rd should be rounded to 8920. Corrected OK Appendix A, page 53

Appendix A, Inhalation Report, Table 1, Page 29. The fourth value for July 24th should be 9960. This was a late

entry. Corrected OK Appendix A, page 55

Appendix A, Inhalation Report, Table 1, page 32. The fourth value for September 30th should be 9890. Corrected OK Appendix A, page 58

Appendix A, Inhalation Report, Table 1, page 34. The

fourth value for Nov. 18th should be 8960. Corrected OK Appendix A, page 60

Appendix A, Inhalation Report, Table 1, page 35. The first value for Dec. 2 should be 11,800. Corrected OK Appendix A, page 61

Appendix A, Inhalation Report, Table 1, page 36. There is a notation made 5 days after exposure for Jan 1, 2002 that there were excess bubbles before the cylinder change, explaining the low nominal value. There were no notations of bubbles anywhere in the CMR. How was this observation

made? Additional footnote added to data to state that original entry added late as observation not

documented at time of observation and that cannot be proven after the fact. OK

Appendix A, Inhalation Report, Table 1, page 38. The third

value taken on Feb. 12 should have been 7110 as this one would more represent the previous 1.5 hours. 9200 was read 3 minutes after an adjustment. Reviewed. As per previous responses, repeat samples were taken (large air bubble during sample) and the average was reported. No adjustments were noted in the data. OK Appendix A, page 64

Appendix A, Inhalation Report, Table 1, page 39. Please

check the second value for March 2nd as it seems that 4640 was more representative of what was in the chamber for the previous 1.5 hours. Reviewed. As per previous responses, repeat samples were taken (air bubbles in flow meter during sample) and average was reported. No adjustments were noted in the data. OK Appendix A, page 65

Appendix A, Inhalation Report, Table 1, page 40. The third value for July 1 st should be 19,900, I believe. This may have

been misread. Corrected OK Appendix A, page 66

Appendix A, Inhalation Report, Table 1, page 50. The second value for the 20th of January should be 20,100. Please verify. Corrected OK Appendix A, page 76

### 211(b) Toxicology Research Group MTBE Two-Generation Reproducti on Toxicity Study Report

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17 5/6/2010 

### Comments Action/Response Page
Appendix A, Inhalation Report, Table 1. There appears to be an inconsistency with how values are reported when a chamber is noted to be too high or too low in concentration.

When such an excursion is noted , the system is adjusted and a reading is taken to verify that the concentration is closer to target. The inconsistency occurs when the additional value is reported, a mean of the two values is reported (mid-dose, 7/9/01), or the original value is reported. It seems that the original value should be reported as this is more representative of what was in the chamber for the previous

1.5 hours. 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 B, Analytical Report, Pages 5 - 7, text table presenting dates of sample receipt and analysis. The following typographical errors and/or omissions were noted:



### a. Samples for Exposure 42 are not included in the text table. These samples were collected and received on July 24, 2001 and were analyzed on July 25-26, 2001.


### b. For Exposure 56, the analytical data indicate that analyses were performed on August 8 and 9, 2001 (not August 7 and 8 as reported).



### c. For Exposure 84, the analytical data indicate that analyses were performed on September 5 and 6, 2001 (not September 4 and 5 as reported).


### d. For Exposure 182, the Charcoal Tube Monitoring Record indicates that samples were collected on December 15, 2001 and deliv ered to Analytical on December 17, 2001. The text table reflects an exposure date of December 17 rather than December 15. Also, Exposure 182 corresponds to the exposure on December 11, 2001. D ecember 15, 2001 was actually Exposure 186.



### e. For Exposures 175 and 182, the Charcoal Tube Monitoring Records indicate that some additional samples were collected between December 4 and 15,

2001. These include an additional control chamber sample (no. 1026-D1) on December 6, 2001 and samples from all chambers (nos. 1027, 2027, 3027 and 4027) on December 11, 2001 (Exposure 182). These additional samples are not included in the text table of samples received and analyzed. Please correct and clarify as appropriate.


### f. For Exposures 224, 231 and 238, the samples were stored for a period of 1 to 15 days from the time of collection to the time of delivery to the analytical laboratory. It is not clear from the data how the samples were stored prior to transfer to the analytical laboratory. Are there stability data to support that the a-e Corrected

Apppend I receipt of samples and analysis must match actual analysis days /r Exposure 182 and 177

There were no reported samples for Exposure 182 but there were reported samples for Exposures 177 and 186. Pages 6 and 36 have

been corrected to be consistent OK

f-g There was no freezer stability data OK

### 211(b) Toxicology Research Group MTBE Two-Generation Reproducti on Toxicity Study Report

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18 5/6/2010 

### Comments Action/Response Page
storage conditions did not impact on the analytical

results?

g. Several of the samples were stored in the freezer for one or more nights prior to analysis. Are there stability data to support that freezer storage does not impact on the analytical results? generated and this will be added to the GLP Statement in the report. Ok

Appendix B, Analytical Report, Tables III-A and III-B. For Benzene, <LOQ should be defined on the table. The analytical data indicates that this value is less than 25% of the area count of the component in the test material standard, whereas elsewhere in this report (e.g., Table III-C), it is defined as less than10%. In addition to table corrections, the

data should be corrected/clari fied to reflect the correct definition of LOQ. Report and data have been clarified. ok

Appendix B, Analytical Report, Tables III-A through IIIZ12. For the coeluting peak for MTBE and 2,3- Dimethylbutane, is there a % that is being used to calculate the estimated relative contribution of MTBE versus 2,3- Dimethylbutane in this peak. Should this be included in the analytical report so that the amount of MTBE in the chamber samples can be better defined? MTBE co-elutes with 2,3-dimethylbutane for all exposure analyses during the analyses and

there is no estimated relative contribution of MTBE versus 2,3-dimethylbutane found in the peak. Added this to the report. ok

Appendix B, Analytical Report, Table III- Z. For the control chamber, results are reported only for the re-sample collected on December 6, 2001 (sample 1026-D1, Exposure 177). The results for the original sample (no. 1026, collected December 4, 2001), which showed isobutene results >LOQ for initial injection and re-injection, are not reported. It is recommended that the rejected results be fully addressed in the analytical report. Rejected results have been added to the report with footnoted explanation. ok

Appendix B, Analytical Report, Table III- Z1. The Table header indicates Exposure 182. However, it appears that the reported results are for samp les collected on December 15, 2001, which was Exposure 186. Corrected. ok

Appendix B, Analytical Report, Table Z1. Charcoal tube samples were collected and analyzed for Exposure 182 (December 11, 2001). No results are reported for these samples. The analytical data indicates that the results for these samples were invalid, but does not provide any specific details as to why they were considered invalid. Additional details should be provided in the study data as to why these

results are invalid and this should also be addressed in the analytical report. Rejected results have been added to the report with footnoted explanation. ok

### 211(b) Toxicology Research Group MTBE Two-Generation Reproducti on Toxicity Study Report

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19 5/6/2010 

### Comments Action/Response Page

Appendix M, F1 Generation, Individual Feed Consumption. Throughout the appendix, a footnote of “IW” is used to denote invalid food consumption weights, which is then further defined in the footnotes as “Feed left greater than

feed fed”. Review of the data indicates that this is an incorrect explanation for some of the “IW” values. (For example, for animal 3007, day 126, the weight was invalid because the animal was in cohabitation at the previous

interval and no feed left value could be recorded. For animals 3006 and 3009, day 133, the values were invalid because the animals were found in each other’s cages.)

Values denoted as “IW” should be reviewed and an accurate explanation for the invalid weights provided in the report appendix. Data and report edited and IW defined as ‘invalid weight’. ok

Appendix P, P0 Generation, Individual Estrous Cycle Data and Individual Mating Determinations. For animal 2562, Day 66, an “M” is reported for the estrous stage. The edit records indicate that the Day 66 estrous stage for this animal was to be changed from “1” to “3” on August 17, 2001. According to information provided to the auditor at the time of this audit, stage 3 corresponds to “E” not “M”. An additional edit appears to be necessary in order to correct this

entry. This edit referred to the data collected on day 65 (premating day 17), not 66 (premating day 18). ok

Appendix P, F1 Generation, Individual Estrous Cycle Data and Individual Mating Determinations. It is not clear how the “Evaluation Days” presented in this appendix were determined for this generation. Can this be clarified in the report? This is now clear in the new format. Not sure what ‘Eval. Days’ were and where confusion was as it has changed. Now for the F1 parameter of ‘# with abnormal cycle’ it is not tallied as P0 was. _ need tally consistently

F1 tallies corrected in Appendix P.

Also corrected on Table 14 OK

Appendix R, F1 Generation, Individual Delivery and Litter

Data. The following items should be addressed:



### a. For “Number of Live Pups”, the first column presented is for Day 0. Should this be Day 1? (see this appendix for P0 generation).


### b. The footnote at the bottom of the page indicates that the Day 4 column is pre-culling. The appendixactually presents two columns for Day 4, one preculling and the other post-culling.



### c. The appendix does not include the number of live pups at Day 28 (day of weaning).

d. For females 1504 and 4525, define abbreviation “pc”, which is used in the “Duration of Gestation” a) Appendix corrected. ok

b) Appendix corrected ok

c) Appendix corrected ok

d) ‘pc’ removed from appendix ok Appendix R, pages 5-8

### 211(b) Toxicology Research Group MTBE Two-Generation Reproducti on Toxicity Study Report

Reviewer Checklist

20 5/6/2010 

### Comments Action/Response Page

column.

Appendix W, P0 Generation, Individual Parental Necropsy Observations. Page 1 (which presents animals 1051 through 1063) was missing from the audited copy of the report. Appendix corrected. ok Appendix W, page 1

Appendix Y, Organ and Final Body Weights, P0 and F1

Generations:

Throughout the appendix, it is recommended that the reasons for missing organ weights be included as footnotes. Currently, the appendix the mi ssing values are left blank with no accompanying explanation. It appears that a notation on the preface page explains. There is no preface page... page 1 is first page of data. Appendix Y, page 1

Appendix Y, F1 Generation, Organ and Final Body Weights. For Group I male 1008, an adrenal weight of 0.6718 grams is recorded in the data and reported in the appendix. This weight is approximately 10 times greater than the adrenal weights for other animals and in the absence of any documented gross enlargement, may represent an erroneous weight. The study director should review the data and make

a decision as to whether this value should be excluded from group calculations and statistics. I signed a memo to delete this erroneous entry from the calculations in the report. Table 22 re-printed excluding 1008 adrenal weight. Footnote added to Appendix Y. ok

Appendix Z, F1 Generation, Individual Pup Organ Weights. No spleen or thymus weights are reported for pup no 4553-5. hese weights were to be added per the edit records dated October 18, 2001. This data was back-entered and re-reviewed and Table 23 and Appendix Z reprinted for the

report. ok

Appendix Z, F2 Generation, Individual Pup Organ Weights. The organ weights for F2 pups are reported to one more decimal place than appears in the raw data printouts contained in the study records. Please address/clarify as appropriate. This is a feature of the TASC software that

cannot be revised. ok

Appendix AA, P0 and F1 Parental Animals, Individual Gross and Microscopic Observations. Several animals had gross observations on the skin (hair thin/absent, etc.) that were not saved at the time of necropsy (not considered significant as per SOP TX05-2). In the appendix, the microscopic observation corresponding to the skin gross observation typically states “No micropathology observations on tissue”, which would seem imply that the tissue was examined and no findings noted when the tissue was actually not examined.

It would be more accurate to indicate that the tissue was not

collected/saved for examination. Affected animals include, “No microscopic observations on tissue” indicates that the pathologist did not examine the tissue. If examined, “Tissue unremarkable” or an abnormality would be presented. This has been footnoted to the preface page. ok

There is no option in Xybion to indicate tissue

not collected/saved. ok Appendix AA

### 211(b) Toxicology Research Group MTBE Two-Generation Reproducti on Toxicity Study Report

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21 5/6/2010 

### Comments Action/Response Page

but are not limited to: 1051, 1071, 1074, 1075, 2061, 3051, 3059, 4051, 4057, 4067, 1551, 1556, 2553, 2562, 2563, 3558, 3571, 4556, 4559, 2017, 4026, 1510, 1518, 4507.

Appendix AA, P0 Parental Animals, Individual Gross and Microscopic Observations. For Group I female 1559, the pathology report indicates that the left kidney was missing at necropsy and was not saved in accordance with protocol requirements. This should be documented as a protocol deviation in the report and study data. According to the data, day 25 females (notpregnant) did not require tissues to be saved at necropsy therefore no tissues should have

been saved for this animal as it was nonpregnant. No deviation needed. ok

Appendix AA, F1 Parental Animals, Individual Gross and Microscopic Observations. For Group I male 1007 and Group III male 3011, the gross observations presented in Appendix AA indicate that the right adrenal was not evident. In Appendix W, (Individual Parental Necropsy Observations), the adrenal observation is reported as “Agenesis, right adrenal”. Shouldn’t the two appendices consistently report this finding? Agreed but these are equivalent terminology and need not be revised. Appendix AA and W match “Agenesis” ok

Appendix AA, F1 Parental Animals, Individual Gross and Microscopic Observations. For Group II male 2019, the lung was apparently missing at necropsy and not available for microscopic evaluation. This should be documented as a protocol deviation in the report and study data. Protocol deviation written and signed by Study Director and added to data and report.

ok

Appendix AA, F1 Parental Animals, Individual Gross and Microscopic Observations. The status for Group IV female

4513 is listed as “F inal Phas e Sacrifice” on the pathology report. The study data indicate that this animal was actually a humane sacrifice due to hindlimb paralysis. Appendix AA should be corrected to reflect the actual disposition for this animal. Applicable summary tables may also be affected. Corrected and Appendix AA reprinted into the

report. ok

B. Additional Raw Data and Protocol Comments Study Protocol, Section 13.1.2.3, Dead and Culled Pups. The protocol states that culled F1 and F2 pups will be examined externally and if unremarkable, will be euthanized and discarded. The study records (Individual Pup Necropsy Observations) appear to indicate that culled pups were examined, at necropsy as an entry of “No Remarkable

Observations” is documented for each culled pup. Please document as protocol deviation or otherwise clarify as appropriate. No deviation per protocol necessary since pups were examined externally at necropsy and if normal (recorded in TASC) were

discarded. No other examinations were done if apparently normal. ok

Study Protocol, Section 13.1.5, Neuropathology Evaluations. The study protocol states that selected pups will be This was documented by memo at time of study and will be included in protocol

### 211(b) Toxicology Research Group MTBE Two-Generation Reproducti on Toxicity Study Report

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22 5/6/2010 

### Comments Action/Response Page
anesthetized with approximately 1 mL/kg of ketamine/xylazine/acepromazine. The study data indicate that the anesthesia dose was increased to approximately 2 mL/kg (per memo) with an additional amount being given if needed. The protocol should have been amended to change

the amount of anesthesia solution. Since an amendment was not prepared, this should now be documented as a protocol deviation in the study data and report. amendment #4 to be provided with the 2nd draft report. ok

Study Protocol, Section 13.1.6, GFAP Evaluation. The protocol states that F1 pups (5/sex/group) not selected for mating will be randomly selected by computer for GFAP evaluation. Since the schedule was changed (protocol amendment 2) so that all GFAP assessments could be performed on a single day (postpartum day 27), the GFAP animals were not really randomly selected from the population as a whole, but rather from the subpopulation of litters born on September 13, 2001. It is recommended that this be clarified in the study data. An explanatory memo added to the study file to clarify that only pu ps on PPD 27 selected

for the GFAP evaluation. ok

Computer Raw Data Change and Additions. Documentation of computer edits indicated that the reason for data corrections and changes was frequently given as “Technician Error” even when it appear ed that there had been a reexamination or re-weigh which resulted in an additional

observation or a different value. It is recommended that reevaluations and re-weighs be documented as such in the edit records to more accurately de scribe the reason for the

corrections. We cannot now edit the reasons that were recorded during the course of this study; however, the suggestion is noted for current

and future studies. ok

Charcoal Tube Monitoring Records and Sample Transfer Documentation. It is not always clear from the sample

documentation where the charcoal tubes were stored if not transferred to the Analytical Department on the day of collection. This should be clarified in the study records. SOP Deviation memo written for absence of documentation of storage location. ok

Analytical Data. For Exposure 182 (samples co llected December 11, 2001), the analytical results were considered to be invalid. Additional samples were then collected on December 15, 2001 (Exposure 186), analyzed and reported. The analytical data does not provide sufficient detail as to why the Day 182

results were considered invalid. It is recommended that this be addressed in greater detail in the study records and also discussed in the final report. Additionally, the control chamber sample collected on December 4, 2001 (Exposure 175, sample 1026)) yielded a “suspicious” result per the analytical data and was re sampled (Sample 1026D-1) on December 6, 2001 and rean alyzed. Again, it is Additional explanation was included in the study data regarding the suspicious results. ok

### 211(b) Toxicology Research Group MTBE Two-Generation Reproducti on Toxicity Study Report

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23 5/6/2010 

### Comments Action/Response Page
recommended that additional explanation be included in the study data and report regarding the suspicious results.

A Deviation Report filed in the study records (dated February 14, 2002) states that one adrenal for animal 2003 was lost during necropsy and adrenal weight therefore not collected. Review of the reported individual animal pathology and organ weight data for this animal do not support that one adrenal was missing (i.e., no comment to that effect is noted in the gross pathology observations and a plausible adrenal weight is reported). The data should be reviewed and the deviation and/or reported results revised as appropriate to reflect actual study events. The animal number was corrected to #1005 on the original deviation and agrees with the

report.
ok

GFAP Sacrifice.

No documentation of the sacrifice of GFAP animals is retained at HLS, (Dr. O’ Callaghan takes the original necropsy records with him). Since HLS personnel contribute to the conduct of these procedures, it is recommended that consideration be given to retaining a copy of these forms at

HLS. Will be considered for any future studies. For this study, the data from Dr. O’Callaghan will be archived at HLS after the final report. ok

Charcoal Tube Sampling Calibration Procedures. The last calibration procedure found in the study records (Volume 2, Book 2 of 36) was dated February 17, 2002. It appears that there should have been one additional calibration done prior to the February 26, 2002 sample collection. The necessary documentation should be added to the study records or the lack of documentation addressed as appropriate. SOP Deviation memo written on 10June04 for addressing the missing calibration on

26Feb02. ok