211(b) Toxicology Research Group 

MTBE Two-Generation Reproduction 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/m3

 for the Gasoline MTBE Vapor Condensate. 

Statistically significant increases in epididymal sperm 

concentration and percent abnormal sperm were observed at 

20,000 mg/m3

 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 historical controls to supplant 

statistically significant findings is inappropriate and would 

respectfully submit that the reproductive NOEL be revised 

downward to 10,000 mg/m3

. 

This issue could be resolved by designating 

20,000mg/m3

 as the NOAEL 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 "tests" 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 Reproduction 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 results. 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. Nonetheless, 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 concentrations 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 Reproduction 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 consumption 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 significant differences with 

treated groups. While 10000 and 

20000mg/m3

 F1 male feed consumption was 

significantly decreased during all 7 weeks, 

statistically significant decreases was seen in 

females exposed to 20000mg/m3

 only at wks 

1, 3 but more frequently at 10000mg/m3

 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 Reproduction 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, namely 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 about using another statistical 

test. 

Control (P0 and F1) and 20000mg/m3

 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 increased 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/m3

 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 between 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 the 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 mean value of abnormal sperm 

was within the range of historical 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 presented 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 Reproduction 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 concluding 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 the 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/m3

 P0 rats is 

only 0.7% greater than controls (2% vs. 1.3%) 

and although statistically significant, falls 

within the historical range 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 significance. 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 Reproduction 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 treatment effects were noted in 

survival. GFAP levels were also 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; microscopic 

finds 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 increased 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/m3

 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/m3

 level of 

exposure. 

No adjustments to the text are considered 

OK_
211(b) Toxicology Research Group 

MTBE Two-Generation Reproduction 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 affected 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/m3

 of gasoline MTBE was concluded. 

I concur with the report conclusions. 

No comment necessary 

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/m3

 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/m3

 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 director 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 investigator 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 Reproduction 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 their 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 4th

line )

Page 7, 1st – 5th 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 macrophage 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 were known as Chevron Research 

and Technology Company. 

Company name corrected. 

21 

OK_
211(b) Toxicology Research Group 

MTBE Two-Generation Reproduction 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. Please delete the second and 

third sentences in this paragraph as unnecessary. 

All done. 

OK_

Page 43, section 3.1, 5th 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 values and the intergroup 

statistical comparison. A later reproduction study had a very 

nice write-up of estrous cyclicity 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 Reproduction 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 occurred 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, 3rd 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 abnormal sperm reported by PAI’s 

Historical Control (0 – 6.7%). As a result, the increase in the 

number of sperm/gram of epididymis 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 toxicological 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 Reproduction 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 row 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 completed, 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 prepared 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 sperm 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 Reproduction 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 and pituitary) were examined 

microscopically from the randomly 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 Reproduction 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 indicate that F1 parental females in 

the 20000 mg/m3

 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 only 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 examined 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 microscopic 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 included as a post- mating death. 

Please correct or clarify 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 Reproduction Toxicity Study Report 

Reviewer Checklist

14 5/6/2010 

Comments Action/Response Page 

page 6. For the males, the total numbers of animals 

terminally sacrificed are reported as 18, 15, 16 and 16 for 

groups 1, II, III and IV, respectively. These numbers do not 

account for all male terminal sacrifices. 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 significance 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 Reproduction Toxicity Study Report 

Reviewer Checklist

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 adjustment 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 after 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 Reproduction Toxicity Study Report 

Reviewer Checklist

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 1st 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 Reproduction Toxicity Study Report 

Reviewer Checklist

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 delivered 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. December 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 Reproduction Toxicity Study Report 

Reviewer Checklist

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/clarified 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 samples 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 Reproduction Toxicity Study Report 

Reviewer Checklist

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 appendix 

actually 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 Reproduction 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 missing 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 Reproduction Toxicity Study Report 

Reviewer Checklist

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 Phase 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 Reproduction Toxicity Study Report 

Reviewer Checklist

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 pups 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 appeared 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 describe 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 collected 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 resampled (Sample 1026D-1) on 

December 6, 2001 and reanalyzed. Again, it is 

Additional explanation was included in the 

study data regarding the suspicious results. ok
211(b) Toxicology Research Group 

MTBE Two-Generation Reproduction Toxicity Study Report 

Reviewer Checklist

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