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

Reviewer Checklist

Comments Action/Response Page

EPA API reviewer response (black) Lab response (red) Plus_

API verification of lab response

In general, the NHEERL reviewers concluded that the

studies were conducted in accordance with the testing guidelines, once changes detailed in the API peer review response are made.

Additional comment is requested on the following points:

- Page 37 notes an extended diestrous period in a significant

number of study animals and abnormal cycle patterns across the study. Page 38 notes that both the 20,000 mg/m 3 and 10,000 mg/m 3 exposure groups exceeded 4 days to mate although significance is attributed only to the delay seen in the 20,000 mg/m 3 group. The results from the 10,000 mg/m 3 group were discounted due to inconsistent cycling of the test group attributed to incidental pseudo pregnancy (defined as extended periods of diestrous) in 9 of 26 animals and 10,000 mg/m 3 was selected as the reproductive NOEL. We are concerned that the reduction of the 10,000 mg/m 3 exposure group to 17 animals will leave insufficient statistical power to allow this determination, and would request that the study group reconfirm the validity of their conclusion after taking the decreased group size into acc ount. Should 17 animals prove insufficient we would request that 2000 mg/m 3 be designated as the reproductive NOEL.

- Note that 22 animals were found pregnant in the 10000mg/m3 group

considered sufficient for statistical evaluation. OK.

Table 14 shows cycle lengths were

evaluated in
23-26 rats/ group. There

were a similar

number that

could be
evaluated for estrus cycles

.



### Peer Reviewer – Schlesinger

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

affected the outcome of the st udy. While the NOEL level for reproductive performance seems reasonable, some consideration should be given to the conclusion for the general NOAEL based upon some of the comments below

. No response needed 

### √

Statistical Analysis: 2.15.1. Is there any reason why two way ANOVA was not used? This would have allowed evaluation of exposure groups at all times for each endpoint. This could be followed by some multiple comparison test. Furthermore, why were tests

conducted at both the 1% and 5% level of significance. The tests and level to be used should be determined a priori before the study begins and only one such level of significance should be used. The computerized statistical testing package employed at the laboratory is designed to perform one-way ANOVA at each time interval and is used for a range of multiple

dose studies. The 1% and 5% levels of significance were pre-selected since they were inherent in the laboratory’s computer statistical package. Comparison at two levels of significance has been common practice in this laboratory. OK

This is similar

to other

studies.

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Comments Action/Response Page

Protocol Deviations: 2.18. There appeared to be a number of errors committed by technicians. While these may not have affected the outcome of the study, they seem to i ndicate a degree of technical "sloppiness" in the conduct of the study. 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 One-Generation Reproduction study is a considerable undertaking and involves a 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 brought this issue to the attention of the laboratory to strive to minimize protocol

deviations. 

### √

Chamber Monitoring: 3.1.

It is noted that there were some differences between the

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

calibration of the chamber airflows and the calibration of the

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

calibration of all monitors and flows are extremely accurate.

There should be no such in accuracy of any atmospheric monitor. In any case, the ta rget, analytical and nominal levels seem to be very consistent.

The investigators need to explain the source of the particles in the exposure atmospheres. Was the incoming chamber air filtered appropriately? Any particles could act as adsorption sites for vapors in the exposure atmosphere. The statement that the "...atmospheres were essentially vapor only" is not quite right. While the vapors may not have produced particles, there were clearly particles in each of the exposure atmospheres. Furthermore, the range of particle sizes was quite large, going from less than 1 micrometer to over 5

micrometers in a number of cases. To be precise, the report stated that the ratio

of the measured to nominal concentration was not 1:1, and speculated that the cause MAY have been due calibration error. However, as Dr. Schlesinger also pointed out, the target,

nominal and measured concentrations were close, varying 2% or less. The RG feels confident that the reported chamber concentrations accurately reflect the atmospheres to which the animals were exposed. With values so close together, the explanation for possible variation from the Results and Summary will be deleted. Deleted and indicated acceptable differences. 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 + TBA 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 34 [first draft] indicates that the average particle

concentrations in all of the chambers including controls were 5-5.7 micrograms/m 3 

### √

» same page in draft 2

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

Reviewer Checklist

Comments Action/Response Page
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. 

### √

Physical Observation Data: 3.2.3. There seems to be an exposure related increase in alopecia. Dr. Schlesinger is correct that the incidence of alopecia of the extremities/snout of male rats increased in an exposure related manner, exhibiting maximum incidence in the mid dose group, and including 3 control rats, by

week 8. Similar effects were not seen in females where the total reported incidence was 1 animal in the mid dose and 2 in the high dose. The study director will be instructed to add these observations to the appropriate

Results section

Added to discussion. 

### √

3.2.2_p. 35

Parturition and Litter Survival Data: 3.2.10. Data in Table 16 suggest a treatment related increase in number of pups dying, missing or cannibalized at the two higher exposure levels that doe s not seem to be clearly noted in the report text. The statistically significant reduced pup viability indices and increased incidence of pups stillborn or dead in the 10000mg/m 3 and 20000mg/m 3 groups were due to two litters in the 10000mg/m 3 group: one dam delivered 10 stillborn pups, and in the other litter all 12 pups died by the day 1 weighing., and to one litter in the 20000mg/m 3 group in which 7 of 17 pups died by day 1 weighing. When these

anomalous litters are removed from calculations, no statistical ly significant effects were observed in the number of pups or pup viability index. Moreover there was no dose response across these levels for pup mortality.

Section 3.2.9 revised to add information on

stillborns. P. 39

OK

When we publish, we

should include the range for

controls in the total program.

.

Postmortem Data: 3.4.1. The lung discoloration in males seems to be treatment related. Perhaps this is meaningful in spite of the lack of any effects on lung weight or histopathology. Dr. Schlesinger is correct that the number of rats with discolored foci in the lungs was observed to increase in an exposure-related manner in both sexes at necropsy of parental animals. As reported in the text, no histopathological effect s such as alveolar macrophage aggregates consistent with discolored foci were seen and no changes in lung wt occurred. Th erefore, the increased frequency of these background lesions is likely a marker of subs tance exposure but is not considered to be of adverse biological significance. HLS_ Consider adding a sentence that these OK

p. 40

3.4.1

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Comments Action/Response Page
lesions are likely a marker of substance exposure not considered adverse.

Added to discussion. 2 n d paragraph

Postmortem Data: 3.4.3. It is not clear why the effect on spleen weight clearly observed in this study is discounted based upon results of the 13-wk study. Statistically significant decreases in absolute and relative spleen wt were seen at all doses in parental females and in 10000mg/m 3 and 20000mg/m 3 parental males. As pointed out by Dr. Goldsworthy, the consistency of these

decreases as well as a similar effect considered signifi cant in pups from 20000mg/m 3 exposed dams make it difficult

to dismiss these observations in the absence of concomitant histopathology.

Revised the discussion. Repro NOEL
and NOAEL p.43

also p.41
section 3.4.3

2 nd paragraph

ok 

### Peer Reviewer – Goldsworthy

The study was scientifically sound and was properly conducted. Exposures were properly conducted and adequate to test the potential reproductive toxicity of Gasoline TBA Vapor Condensate. None of the protocol deviations noted would be expected to adversely effect study results or data integrity. Protocol deviations listed on page 32 did not note the temperature or humidity excursions outside the specified range. No response needed 

### √

No effect of treatment was noted on survival, clinical observations, feed consumptio n, estrus cycles, mating, fertility or gestation indices, or on other reproductive parameters up to parturition except for animals in the high dose group which took slightly longer to mate as compared to other groups. There were exposure related decreases in body weights (Table 5) and body weight gain (Table 6) in the 20,000 mg/m3 animals during the pre-mating period. This is not correctly stated in Summary (page 6) and Conclusion (page 42) and should be modified. No treatment differences were observed with pregnancy rates, duration of gestation and percent of females completing delivery. No

differences between treated and control groups were noted in all parturition parameters, including total number of pups delivered, viability and lactation indices, the number of implantation sites per litter, the pup sex ratio, and the number of live pups per litter. Additionally, sperm count, motility, morphology and caudal epididymis weight were not altered by treatment. Dr. Goldsworthy’s comm ent is correct. The Conclusions and Summary will be revised to include decreases in absolute body weight as well as decrease in weight gain Group 4 absolute body weights were not decreased but gained less than the controls.

Discussion OK as is. 

### √

There were no exposure-related differences in macroscopic postmortem assessments. Increased kidney weights were observed in male rats (parental generation) at the two highest Statistically significant decreases in absolute and relative spleen wt were seen at all doses in parental females and in 10000mg/m 3 and

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Reviewer Checklist

Comments Action/Response Page
exposure levels; this is consistent with the expected induction of hydrocarbon nephropathy. Microscopic evaluation was not performed to confirm this observation. Hydrocarbon nephropathy is generally not considered to be relevant for human risk assessment. Statistically significant decreases were seen in spleen weights at all exposure levels

in both males and females. These decreases were seen in absolute values as well as relative values to body and brain weight. This finding was di smissed as adverse because similar effects were not observed in a previous 13-week study. However, data findings must place emphasis within

the context of curren t study control values and results. The consistency of splenic weight decreases in all dose groups, in both sexes as well as observed in pups, suggests that this finding may be an adverse finding. Unfortunately, histopathology results were not conducted to clarify this finding. Without further clarification as to the relevance of the data, this report should note the decreased spleen weights in Summary and Conclusion sections. These data also will affect the NOAELNOEL setting. 20000mg/m 3 parental males. As pointed out by Dr. Goldsworthy, the consistency of these

decreases as well as a similar effect considered signifi cant in pups from 20000mg/m 3 exposed dams make it difficult

to dismiss these observations in the absence of concomitant histopathology. Summary and R&D revised to note this spleen weight effect. Summary p7 paragraph 1

p41

3.4.3 Paragraph 2

OK

As noted above, kidney, liver or spleens were not evaluated histopathologically. It is not clear why only these three organs (plus brain) were not examined microscopically as were all other tissues listed in Table I (page 26); this lessens the strength of the report. Both gasoline and TBA have been shown to induce a variety of renal and hepatic alterations. This study was designed to evaluate reproductive effects. The RG decided that since extensive histopathology had been performed in the 13-week study and results documented on a variety of organs, it was not necessary to repeat here. 

### √

There were no exposure-related differences in clinical observations or macroscopic postmortem evaluations in lactating pups from air control animals. Table 17 and 18 demonstrated statistically significant decreased body weight and body weight gain at some time intervals in the high dose

groups. Thus, the first sentence in section 3.3.2 and a similar sentence in Summary section (page 7) should be revised and these findings appropriately note d. Significant decreases in spleen weights were seen in the 20,000 mg/m 3 exposure level animals as compared to controls. The Conclusions and Summary will be revised to include decreases in absolute body weight as well as decrease in weight gain.

Summary and R&D sections 3.3.2 and

Conclusions revised. 

### √

In summary, test animals were properly exposed to adequately test the potential reproductive toxicity of gasoline-TBA. A NOAEL of 10,000 mg/m 3 was determined; however, this NOAEL setting doe s not take into account the decreased spleen weights observed at all exposure levels. With respect to reproductiv e performance, the NOAEL setting of 10,000 mg/m 3 is warranted. Done to reflect that “...with respect to reproductive performance, a no observable effect level (NOEL) of 10000 mg/m3 of Gasoline TBA Vapor Condensate was also concluded with an NOAEL of 20000 mg/m3.” OK



### 211(b) Research Group Reviewer (Roberts)

General Comments

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Reviewer Checklist

Comments Action/Response Page
The report should have sequential page numbering from start

to finish.

All of the reports should have an appendix summarizing historical control data for reproduction endpoints. This should be included in all of the reports. This doesn't need to be massive; endpoints such as fertility and gestation indices, litter size, sperm counts/motility, etc., are the types of endpoints that would be of most value. HLS and PAI HCD added to the report as Appendix EE. OK

The handling and write-up of all of the estrus (vaginal smearing) data was very nice, with good explanation and the periods of pseudopregnancy were excluded from calculations. This is how all of the reproduction studies in the 211(b) program should be handled. No response needed

There are still issues with classification of stillborn and found dead pups. Individual data in the appendices and the summary tables do not seem to add up with consistency. Tables and appendices reviewed and corrected

as necessary. OK

Specific Comments P. 2, Statement of Compliance. "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." Not only should this be completed, but most likely it is being included in the SOT poster. This needs to be rectified between API and HLS. Corrected 

### √

P 2

P. 5, 4th paragraph. Please replace "reasonably close" with "acceptably close,"

since there should be criteri a for satisfactor y comparison. Revised to “acceptably close, in the opinion of the study director,” P6 1st line
OK

Please consider deleting the sentence beginning, "The exact

cause of the differences.. .. " since this is unnecessary. Deleted. 

### √

P 6, Parental Organ Weights. Please see comments for p. 41, parental organ weights. Added to R&D but not of value to this discussion in the Summary. 

### √

P. 15, 2.4.1, Supplier.

Please correct the name to ChevronTexaco Research and Technology Company, or ChevronTexaco Global Technology Company, whichever will be correct to QA and consistent throughout the 211(b) program. Revised to: ”Chevron Global Technology Services Company “ for consistency with other reports. 

### √

P. 16, 2.4.9, Dispensing. Revised to: ”Chevron Global Technology Services Company “ for consistency with 

### √

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Reviewer Checklist

Comments Action/Response Page

Please correct the name to ChevronTexaco Research and Technology Company, or ChevronTexaco Global Technology Company, whichever will be correct to QA and consistent throughout the 211(b) program. other reports.

2.4.1 0, Archival Sample. The wording suggests that the ar chival sample is stored as described in 2.4.8; i.e., in an outdoor solvent shed or indoor laboratory. Please clarify. Clarified to indoor lab. 

### √

P. 19, 2.9.4, Feed Analysis.

The comma after "feed" is incorrect. Corrected. 

### √

P. 20, 2.9.9, Environmental Conditions. The actual low temperature, 14.3 C is very low compared to the intended range. Please in clude when this happened (study day/phase) and how long it persisted until the room temperature was within the desired temperature range. This excursion was on 26Sept02 and

recovered to 18C within 1 hour. I have added to report that “Excursions outside the specified range were generally brief in

duration and considered not to have affected the integrity of the study.” 

### √

P. 22, 2.1 1.2, Detailed Physical Examinations.

The comma after "as well as" is incorrect Corrected. 

### √

P21 bottom

P. 32, Protocol Deviations.

- No. 1: What is meant by "n o definite effect?" My

recollection is that, after this occurred, a number of the female rats in estrus persisted in estrus longer than usual.

Please check.

- No. 3: If pup No. 5 was not preserved, what was the

consequence? Were less than 3/sex preserved, or was another pup preserved in its place?

- No. 5: Please reword; the co rrect wording suggests that

technician error caused No. 1527 to be found dead.

- No. 6: Were efforts made to contact the supplier and

obtain the appropriate bedd ing analyses? Example analyses from 1999 are not very relevant (and if this occurred in earlier reports, I apologize for not catching this problem). No. 1. revised to no ‘apparent’ effect.

No. 3 No consequence: Only macroscopic examination was required for 3/sex at terminal sacrifice (Day 28); no preservation was required. Only pups with abnormalities were required to be preserved. This pup was found dead on Day 4. Ther e was no milk in the stomach, therefore it should have been preserved

.

No. 5. Reworded for clarity.

No. 6. Further contact with the vendor (Fisher Feed) was made on 11Sept09 and they

confirmed that actual lot numbers for the bedding for these studies were not available. Therefore, the exampl e analysis already

included should be sufficient. 

### √

OK

P. 33, 3.1, Chamber monitoring. Table 6 in Appendix A is for the testing room, not the chambers. Could you please point out where the chamber oxygen levels are located? Table 6, week 17, indicates 19% oxygen for Group 1, so if this is the table that represents chamber oxygen levels, the text on p. 33 needs to be

corrected from "at least 20%" to "at least 19%." Table 6 in Appendix A clarified for the room

and chambers results. Text on p. 33 corrected from "at least 20%" to

"at least 19%." 

### √

P173 / 654

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Comments Action/Response Page
P. 34, 3.1, last paragraph. Please revise "reasonably close" to "acceptably close," since

there must be a criteria fo r satisfactory comparison. Revised to “acceptably close, in the opinion of the study director,” P33

OK

Last sentence.

Grammatical correction: "data was" should be "data were." Corrected. 

### √

P. 35, 3.2.1, Mortality.

Table 1 shows that a Group 2 female (2549) died/sac's postpartum and two Group 3 females (3536, 3548) died/sac'd postpartum. This is not discussed in this section or tied in elsewhere, such as in 3.2.1 0 for litter survival (not tied in for the dams). The listings in Table 1 need to be explained, if only to clarify why the animals were sacrificed. Also, please read comments directly below for 3.2.3. Added to section 3.2.1.

No change to Table 1. This is a summary table. Explanations for individual animals can be found in Appendix C. 

### √

P35

3.2.2. Doesn't exist. Please correct the section ordering. Section ordering corrected. 

### √

3.2.3, Physical observations. P35

Table 3 does not seem to match Table 1 for numbers of animals with terminal sacrifice during gestation. This needs to be explained and/or correct ed. Also, the row for "found dead" in Table 4 does not appear to concur with Table 1.

The fate of animals that did not survive to scheduled termination needs to be explained in a clear manner so that a

reader will understand what ha ppened without needing to dig through the individual data tables. With the exception of animals with Total Litter Loss, Table 1 does not show Terminal Sacrifices. No change to Table 1 with regard to Terminal Sacrifices during Gestation (Table 3). A notation was added to show that

animals with Total Litter Loss are included in

the animals that died/w ere sacrificed postpartum. These are classified as Terminal Sacrifices in Table 4 (and Appendix C). OK

3.2.4, Body Weight. Didn't males at 20K also have lower body weight gain during the week prior to exposure initiation? Also, there were additional weeks, beyond what is mentioned in the text, with lower body weight gain. Section revised. OK

P. 36, 3.2.5, Maternal Body Weights - Gestation and

Lactation. Please consider revising this section. The text should focus upon what the findings are. It is apparent from the data that there were no differences in ge station body weight gain; this

should be stated. It isn't clear in this section if there is an interpretation that biologically or toxicologically significant differences occurred in lactational body weight gain; did a prior body weight difference resolve during lactation (i.e., the adverse effect ended), or did the dams put on more weight at the expense of the pups (separate adverse effect)? Section revised.

OK

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Comments Action/Response Page

3.2.6, Feed Consumption. Please see question below related to Table 11. See response to Table 11 comment below. OK

P. 37, 3.2.8, Estrous Cycle Data. This is a nice write-up and explains the results very well. I think that the final sentence, "Adequate assessment of cyclicity was possible despite the incidental episodes of pseudopregnancy" could be strengthened even more, since adequate assessment was not only possible, but occurred. Section revised. OK

P. 38, 3.2.9, Mating, Fertility, and Gestation Indices. This, also, is a nice write-up. There is still some ambiguity about the delay in mating at 20K; although it appears that this is attributed to exposure, it isn't clear what the "normal" range for days to mate is, or how frequently animals miss their first opportunity to mate. Although this was not an endpoint for adverse findings in other studies in our program, the argument for no effects at 10K (artifact of pseudopregnancy) should be consistent with what was seen in control groups (i.e., BIMTBE) with pseudopregnancies. Section revised. OK

3.2.10, Parturition and Litter Survival Data. There are discrepancies between Table 16 and Appendices R, S, U and W. Table 16 lists 21 animals with liveborn, but Appendix R, p. 1, lists 20. Did this eliminate No. 1527, the female that died on LD 1 with one fetus inside? Appendix R lists 21 animals with liveborn. The table and the appendix need to be consistent, and the situation for No. 1527 should be described. Also, Table 16 does not report the same numbers as Appendix S. Table 16 reports that the control group had one litter with stillborn pups, yet Appendices S, U, and W show 2 litters13 pups that were stillborn. To add to the confusion, the two stillborn pups for No. 1527 that are confirmed stillborn by the lung floatation test were reported to have milk in their stomachs. Something is wrong here and needs to be straightened out and clarified before the results can be published. Animal No. 1527 is excluded from Table 16 and Appendix R (delivery and litter data) and Table 18 and Appendix R U (pup weights). Notations were added to the appendices indicating the exclusion.

Since No. 1527 is excluded from Table 16, the pups (Appendix S, U and X) from that litter are also excluded – leaving 1 litter with 1 stillborn pup.

Animal No. 1527 was not excluded from Table 19 or Appendix W (parental necropsy) or Table 20 or Appendix X (pup necropsy).

Notation added to Appendix X indicating that the milk in stomach observation supersedes the lung floatation test, therefore the status for these pups was corrected to “Dead”. Now 3.2.9 Append R _p336 Append U p 388

Append S p340 Append U p384 Append X p 435

append W p424 Append X p435



### OK

P. 39, 3.2.10, 9 Parturition and Litter Survival, cont.. It seems appropriate to incl ude the reasons why total and partial litter loss at 10K an d 20K, respectively, are considered incidental ("occurrences that were considered incidental because...") Section revised. 

### OK

3.3.2, Pup Body Weight.

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Comments Action/Response Page
When discussing the slightly greater pup weight gain at 2K, please consider revising to include comment about the slightly smaller mean litter size that occurred in this group compared to control. Also, there are an excessive numb er of weight gain intervals shown on Table 18. Please consider revising Table 18 to include only those intervals that seem most appropriate, then be consistent across all the studies for the 211(b) program. Section revised.

Table 18 - Intervals cannot be changed due to TASC program limitations. 

### √

√

P. 40, 3.4.1, Postmortem Data, Terminal Estrous. Please consider using a sing le consistent spelling for estrus/estrous. At the end of the first paragraph, please consider adding in the lab hist orical control range for estrus at termination; it will no doubt provide ample evidence of the variability of the endpoint. Spelling OK as is since estrous is adjective

and estrus is noun.

Discussion of the estrus HCD added to the

text. OK



### √

Second paragraph. The criteria for discounting lung discoloration as unrelated to treatment are not convincing. The justification may be adequate for a determination that it isn't adverse, but both males and females had a higher incidence of discolored lungs, and the data look related to exposure. In males, discolored thymus also appears to be increased at 20K; perhaps comparison to control ranges would be appropriate. Discussion of lung discoloration has been expanded. Thymic discoloration (red) is a common finding in rats and is due to agonal hemorrhage (caused at the time of death by the method of euthanasia). It is generally not recorded microscopically by the pathologist since it is considered a post mortem artifact. Since the incidence was so low and only present in males, it is not considered necessary to discuss the artifactual nature of the finding

in the text. OK

3.4.2, Macroscopic Evaluations, Pups. Please see comments below for Table 20 and Appendix X. This section should discuss each section (died, stillborn, culled, necropsied) separately, even if only as a single

sentence each. Discussion has been revised. OK

P. 41, 3.4.3, Organ Weights, Parents.

There should be a comment ab out the increased organ/body weight ratios due to reduced body weight. Mention added at end of this section. P41 end /p 42

1 st

sentence
OK

The argument that the decreases in spleen weight are unrelated to exposure on the basis of the subtonic results is problematic. The subchronic study did not expose the animals for as long, and exposed only 5 d/w. Discussion has been revised. P41 end

OK

Table 1, Summary of Survival and Pregnancy. This table is confusing, since the total females died/sacrificed does not clearly add up to the numbers shown above. Please clarify or correct. No correction necessary. Total females

died/sacrificed is a sum of the categories presented above it on the table. OK

Table 3, Terminal Sacrifice. The numbers don't seem to agree with Table 1. Only unscheduled deaths are included on Table 1. No change. OK

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Comments Action/Response Page

Table 4, Found Dead. Again, the numbers don't readily add up. This might be handled by discussion in section 3.2.1 . Missing terminal sacrifices added. OK ( died post partum)

Table 11, day 56. Why is the value for group 2 (X=60, SD = 2.6, N=26) statistically significant from control (X=62, SD = 3.5, N=26), but the value for group 3 (X=60, SD = 2.3, N=26) is not? Please check; perhaps this is due to rounding. This is due to rounding. OK

Table 16, Summary of Delivery and Litter.

Please see comments above for sec tion 3.2.10. In the control group, there is one less female with liveborn than females pregnant. Yet review of the individual data in the appendices (R) clearly shows 21 litters with liveborn. I

assume that No. 1527 was not included, but she met the criteria for inclusion in the gestation index.

Again in the controls, only one litter is listed as having any stillborn, yet there were two litters13 pups that are listed in appendices as status stillborn. For one of those litters, the two stillborn pups should be classified as stillborn on the basis of the lung floatation test, but observations for them included milk in stomach (see comments under appendix X).

This doesn't make sense. These data need to be reviewed and the individual data need to tally up to the values presented in summary tables. See response to section 3.2.10, Parturition and

Litter Survival Data comment. P35
OK

Table 20. This table has four page ones; the pagination should be continuous, start to finish. The section of the table that is headed, "Summary of Pups Stillborn Observations" includes a total of 7 pups that the lung floatation test determined to be "found dead," not stillborn. Why are they still on this table? For "Summary of Pups Cannibalized," there are 5 pups in group 3 for whom milk in stomach was undetermined; does this mean that there was one pup for which milk in stomach was determined? If so, it should be listed, but isn't. Entire report will be paginated beginning to

end at finalization.

Statuses for pups corrected based on results of lung floatation tests.

Per Appendix X, no observations for milk

were made for 3547-2. Table 20 is correct as

is. OK

( note: 6 pups &

P456

3547-2 cannibalised)

Appendix A, p. 7, 2.3.2. Please correct the grammatical errors in the second sentence. Corrected 

### √

Appendix X. Some cannibalized pups are listed as status Z for cannibalized, others as stillborn. Why the difference, and If stillborn or dead status could be determined for a cannibalized pup (i.e. a lung floatation test could be performed or observations for milk in the stomach could be made), that OK

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Reviewer Checklist

Comments Action/Response Page
what is the justification for the difference? status is listed. Otherwise, the pup is listed as cannibalized (Z).

For 1527/1&2, both are listed as stillborn, yet have milk in stomach. This doesn't seem to be a likely assessment. Pups will be considered found dead (milk observation overrides lung floatation). Animals footnoted in Appendix X. (Note: p435 Footnoted)
OK

For 252612, the status is S for stillborn, yet the observations indicate milk in stomach-found dead. Why is this listed in a conflicting manner? 1, 2 were two different pups. One was

stillborn and one was found dead. OK

For 253911, the status is stillborn, but the observations indicate that the pup should be classified as found dead on the basis of the lung floatation test. The same is true for 254311, 352811 (which also has milk in stomach), 353617&10, and 453811. 2539-1, 2543-1, 3528-1, 3536-7, 10, 4538-1 –

all were found dead according to data. 3536 – was partially cannibalized meaning it

was undetermined.

Statuses for pups corrected based on results of lung floatation tests. OK



### 211(b) Research Group Reviewer (Cruzan)

p. 6. Summary says there was no effect on body weight - High dose males had significantly lower BW except day 7 and 42; high dose females except day 7, 28, and 42. Discussion of body weights has been revised. 

### √

P6 paragraph 2

p. 38, Section 3.2.9. Was the difference in mating length statistically significant? No 

### √

It seems inconsistent to write-off decreased spleen weights in parental animals as not treatment-related, but decreased spleen weights in offspring as indicative of toxicity. It appears that in both cases, th ere was no histopathological difference in the high-dose. This was used as justification to dismiss in parents but not pups. Discussion of spleen weights has been

revised. P43
OK

Overall, it seems the lab is really reaching to declare a reproductive effect at 20,000 mg/m 3 . They cite a slight, but not significant, extension of the time to pregnancy (2.7+/-1.6 vs. 4.0+/-2.7 days) and a decreased spleen weight without pathology. NOEL of 10000 with NOAEL of 20000 revised in Summary and R&D conclusion. P43
OK



### 211(b) Research Group QA/QC Reviewer
Study Title: Gasoline TBA Vapor Condensate: A OneGeneration Whole-Body Inhalation Reproduction Toxicity Study in Rats

The following items requi re further consideration:

A. Draft Final Report and Associated Raw Data 1–Statement of Compliance, Pa ge, 2: The second sentence Corrected OK

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Comments Action/Response Page
should read, “It was the Sponsor’s responsibility to maintain the method of synthesis, fabrication, or derivation of the test fuel, and was not completed prior to study initiation, but has subsequently been completed.

2–Quality Assurance Statement, Page 4: The protocol and draft report indicate that the Testing Facility was responsible for the GLP compliance of th e subcontractor, PAI. The inspections/audits conducted to ascertain their compliance status should be listed on the Quality Assurance Statement of this report. QA Statement updated to reflect the PAI inspection. OK

3–Page 6, Summary, second paragr aph: It is indicated that

there were no effects of treat ment on body weights during the pre-mating period. It appears that 14 out of 18 body weight intervals had high-dose male body weights statistically significantly re duced when compared to controls. Approximately one-third of the female high dose body weight values were statistically significantly reduced compared to controls. Please clarify and add to ‘conclusion in third paragraph’.

The next sentence indicates th at body weight gains were decreased in an exposure-related manner, yet, in high-dose females only two intervals were statistically significantly reduced. This should probably be indicated. Discussion of body weights has been revised. OK

4–Page 7, Summary, first paragraph: It is indicated that no test substance related microscopic changes were noted in any tissues. It seems the kidney findings should be mentioned even if considered accepted and not relevant. The kidneys were not evaluated in this study; mention was only made of findings in the 13 weeks study. OK

5–Page 20, Environmental Conditions: After review of the data, it is not clear whether indicating that the temperature and humidity were maintained within the specified ranges to the maximum extent possible is true. There are many instances where temperatures far outside of acceptable ranges were noted and maintenance was not called (8/25 and 26 and 9/26; temperatures of 15.8, 15.4 and 14.3, respectively)(during 9/02 there were 11 high readings of humidity over 90% with no maintenance calls). This appears

to be an SOP deviation and should be documented as such. Per REES database records, the temperature alarm on 8/25 and 8/26 was answered by

maintenance within minutes of the alarm and the alarm on 9/26 was answered by ToxOps (EW) who performed a calibration of the probe.

The humidity excursions during September

were all brief and intermittent. None lasted long enough to trigger an alarm. OK

6–Page 20, Temperature: The actual range is 14.3-26.0 degrees C, I believe. Corrected OK

7–Page 20, Temperature and Hu midity: The frequency and

duration of the excursions from desired levels should be given. On most days the average is within the desired range, but for extended periods, the highs and/or lows are out of the desired range. For example, between 9/1 and 26, the relative humidity was above 70% at least once each day for all but 5 days. There are too many brief excursions to list in the report but all are in the raw data. I have added to report for both temperature and humidity that “Excursions outside the specified range were generally brief in

duration and considered not to have affected the integrity of the study.” OK

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Comments Action/Response Page

8–As well, there are times when calls to maintenance were made and these calls were not answered for long periods of time (12/24 maintenance called for a Humidity measurement out of range; the call was reco rded on 1/2/03; the call was answered on 1/15/03). Per REES database records, all calls were answered on the day of the alarm. Paperwork was completed at a later date. In the specific example, the alarm was triggered and answered on 12/24/02, the form was filled out on 1/2/03 and signed by the responding

HVAC technician on 1/15/03. OK

9–Page 32, Protocol Deviatio ns; Deviations numbered 3 and 4 were not found written up as deviations with others in the

data book. Deviation memos are in the data book. OK

10–Page 33, Chamber Monitoring: The oxygen levels were

at least 19%. Corrected. OK

11–Page 35, Physical Observations; Alopecia might beconsidered treatment-related in the males during the premating period. Alopecia added to the discussion. OK

12–Page 35, Body Weights: It is indicated that both males and females showed slightly reduced weight gain for the first 3 weeks of exposure at the high dose level which continued until termination. As noted in item 3 of this report, body weight measurements as well as body weight gains were statistically significantly affected. The text indicating slightly reduced weight gain may be misleading as 10 out of 17 measurements for the males were statistically significant, many reaching significance at the p<0.01 level. Females

didn’t seem to be as affected as the males, so maybe it would be more accurate to talk about them separately. As well, the high-dose males had statistically significantly reduced body weight gain the week before exposures began, so this may

need to be discussed. Revised to emphasize more the difference for

the males and that the differences were statistically significant. OK

13–Page 36, Maternal Body Weights: The deficit in female body weight at the high dose appears to be about 5% at the end of the pre-mating period, and then ranges from 3-5 percent throughout the gestation and lactation periods, ending at about 1.5 percent at LD28 (resolving only after LD 21). Section revised. OK

14–Table 11, Mean Feed Consumption: There appear several suspicious indications of statistical significance. These are – Males, Day 56, Levels 2 and 3; Males, Day 13, Level 2; Females, Day 35, levels 2 and 4. Please verify that appropriate statistical progra ms were run and reported. Correct as reported. OK

15–Page 32, Deviation number 7: Shouldn’t animal number 1529 be added to this list not observed on Day 105?? No correction necessary . Animal No. 1529 delivered a litter on Day 99. See lactation observations (Appendix F) for subsequent

observations. OK

16–Page 37, Estrous Cycle Data: The second paragraph indicates that there were 3 instances of pseudopregnancy in Discussion added for Animal 1529 not 1528. OK

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Comments Action/Response Page
the control group. In Append ix P, only two instances are noted and excluded from the cycle calculation. Should the extended period of diestrous for Animal 1528 also be

excluded?

17–Page 37, Estrous Cycle Data: The second paragraph indicates that there were 9 instances of pseudopregnancy in group 3, yet Appendix P only footnotes 6 extended periods of diestrous that are excluded from the cycling calculation. Shouldn’t animals 3541, 3542 and 3550 have the periods of pseudopregnancy excluded from the cycling calculation? Table 14 will change if these cycle lengths are changed. Discussion revised to 8 instances in Group 3

and 3541 and 3550 excluded and Table 14 and Appendix P updated. OK

18–Page 38, Mating, Fertility, and Gestation Indices: Please verify the group 4 data presented in the text box. I cannot verify the ‘Number missing first opportunity to mate or missing 2 or more opportunities’. Upon further review, we corrected the ‘Number missing first opportunity to mate’ to 7 and concur with 1 for ‘missing 2 or more opportunities’. OK

19–Page 40, Macroscopic Evaluations: Lung observations were not really scattered as indicated in this section. They were actually dose related. See previous discussion of this topic. OK

20–Page 41, Histopathological Evaluations, Parental Generation: Chronic inflammation of the lung appeared slightly increased in the high-dose male animals. Minimal subacute/chronic inflammation is a common finding in lungs of rats on inhalation studies. In the absence of any other findings,

this is not considered treatment related. OK

21–Table 1, No. of Females Cohabited without evidence of mating, for Group 1 should be 3 (#1529, 1548, 1050), with one being pregnant. Please verify. Corrected. OK

22–Table 1, No of Females Cohabited without evidence of mating, for Group 4 should be 2 (4541, 4547), with one being pregnant. Please verify. Corrected. OK

23–Table 1, Pregnant, died/sacrificed: shouldn’t this be 0 as the dam that died, did so on LD1 and should be included in the category, ‘died/sacrificed post partum. Status corrected. OK

24–Table 3, Summary of Clinical Observations During

Gestation: Terminal Sacrifice. It is unclear what animals were part of a Terminal Sac during Gestation. No animals died or were sacrificed during Gestation. Animals that did not deliver a litter were

euthanized on GD 25 as Terminal Sacrifice. OK

25–Table 4, Terminal Sacrifice: For group 1, shouldn’t this

be 20/20? Missing terminal sacrifices added. OK

26–Table15, Summary of Cohabitation Data: Females with defined day 0 of Gestation: These values include females for which there is a day 0 of gestation but were not pregnant.

Should that be the case? Yes, that is correct. OK

27–Table 16: The category ‘Females Completing Delivery Statuses corrected. OK

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Comments Action/Response Page

with Stillborn’ will need to be revised after all of the stillborn/dead/cannibalized/ autolyzed are sorted out.

28–Table 16, Duration of Gestation: Appendix R indicates an N of 20 for Group 1 while this table indicates 19. Please clarify. Corrected in a previous comment. OK

29–Table 16: It appears that the female that died after parturition (in Group 1) is excluded from the ‘females with liveborn gestation index’, ‘females completing delivery’,

‘litters with liveborn pups’, ‘implantation sites’, and ‘live pups/litter’ . It seems that if she lived through delivery, she and her pups could be included in these parameters. No change to table. Exclusion from this table is “all or nothing”. OK

30–Table 16, Pups dying, Missing and/or Cannibalized’ on Day 0 will change when these are sorted out. Statuses for pups corrected based on results of lung floatation tests. OK

31–Table 16, Page 2, Pups Liveborn, not culled prior to Day 21: I could not verify these values and I am not sure what they represent. I see 199, 196, 194 and 219 pups for this parameter in the Group 1, 2, 3, and 4’s respectively.) Values appear to be correct as is: Liveborn minus those culled on Day 4 = Liveborn, not culled prior to Day 21. Values cited are the Pups Surviving 21 Days: Liveborn, not culled prior to Day 21 minus pups dying, missing, and/or cannibalized days

0-21. OK OK

32–Table 16, Page 2, Pups dying, missing, and/or cannibalized: For group 4, Appendix 4 indicates 13, and 12. Please clarify. No change. Per Appendix S, 12 pups died or were missing on Days 0 to 4 and one on Day 5. Table 16 indicates 12 pups during Days 0-4 and 13 pups for Days 0-21. OK

33–Table 20: All pages of this table are numbered 1. All of these tables listed as Table 20 have different headings. They should be separated. All of Table 20 is pup necropsy findings, with the table further broken down by type of death (dead, stillborn, culled, etc), therefore this

should remain as one table. Consecutive pagination will be applied at report

finalization. OK

34–Table 20, Stillborn vs. Dead Pups: According to Appendix S, there are several dead pups that are being designated as still born. These are: Group 1 #1527-1 and 2, lungs do not float, but there was milk in the stomach. Group 2 #2526-2, lungs float and there was milk in the stomach; #2539-1, lungs float; #2543-1, lungs float; #3528-1, lungs float; #3536-7,10, lungs float; #4538-1, lungs float. See response to section 3.2.10, Parturition and

Litter Survival Data comment.

Statuses for other pups corrected based on results of lung floatation tests. OK

35–Since all pups dying or stillborn were reported as stillborn, it is unclear whether the designation of cannibalized, autolyzed and dead pups is consistent. Presently all cannibalized and autolyzed pups on Day 0 are reported as Stillborn. Is this how they should be reported? For example, Animals #2540-1, 3536-1, -2, were listed as stillborn, but were partially cannibalized, so no lung floatation was done, and it was unknown whether there was milk in the stomach. Should these be designated as Z, cannibalized, or Dead? Animal #2528-1, 3533-1, and 3536- Statuses for 2540-1 and 3536-1 & 2 changed

to Z-cannibalized. Dead or stillborn could not

be determined.

Statuses for 2528-1, 3533-1 and 3536-9 are OK as reported. Default is stillborn. Since no determination could be made due to autolysis, pup status remains as stillborn. OK

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Comments Action/Response Page
9 were autolyzed with no lung floatation or stomach observations done. Should these be designated as stillborn

or dead

36–Animal #3547-1 is designated as stillborn in the data, indicating that lungs did not float, and there was no milk in the stomach, but then there is a designation of partially cannibalized with only the head present on Day 1 of Lactation (do I have this confused with pup 2?) Please clarify. 3547-1 = still born on LD0 3547-2 = partially cannibalized on LD1 OK

37–Animal 4532-8 is listed as D, but partially cannibalized on Day 1 of lactation. No lung floatation could be done, should it be classified as Z, cannibalized for consistency?

There should be some kind of explanation (preface page) of how all of these pup deaths are going to be

+summarized so the reader understands how the designations were assigned

. The pup was cannibalized and autolyzed so no exam performed. Per litter observations, (Appendix R), this pup was alive on Day 0, so lung floatation was not necessary on Day 1. Status of D-dead is

correct. Designation of pup death status seems to be self-evident: D = found dead, S = stillborn, Z = cannibalized (dead/stillborn determination not possible) or U = undetermined. OK

38–Table 22, Summary of Pup Organ Weight Data: For the

tissue measurements of the comb ined sexes, shouldn’t the N be 40, not 20, for controls for example? This table probably took a mean of the male and female weights and then averaged them? No change to Table 22. Data presented are litter averages. OK

39–Appendix A, page 46, Injection Volume 86 ul: Acceptable Absorbance Rang e is 0.279 not 0.278. Corrected. OK

40–Appendix B, page 6, last sentence: The extraction efficiency evaluation data and results from 00-6131 should

be included here. Added. OK

41–Appendix B, page 25, TMStd2(Reinj): the reason for reinjection should be given. Injection error reason added to footnote. OK

42–Appendix K, Computer correction data sheet indicates that there should be a change for Animal 2546, LD ? BW. It

is LD 7 and this should be added to the correction sheet. The

incorrect value was corrected. The data sheet has been updated to indicate

that it was LD7. OK

43–Appendix K, Individual Body Weight Gain Data was not presented for the males. Added. OK

44–Appendix M, Ind. Feed Consumption: Animal 1534 and 2540 had ‘invalid feeder weights’ recorded in the data for Study Day 69, yet there is a food consumption for these animals on this day in Appendix M. Could these animals have just been found pregnant and this ‘invalid feeder’ weight was not needed anyway? Please clarify. The TASC data has been edited to reflect the documentation of ‘spilled feeder’ for this study day (as per invalid feeder record).

Table 11/Appendix M reprinted. OK

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45–Appendix P, see item numbers 16 and 17 for additional pseudopregnancies. Appendix P revised. OK

46–Appendix U: Once the S, D, Z designations are sorted out, this appendix will need to reflect corrections. Appendix U re-printed. OK

47–Appendix V, Individual Terminal Estrous

Determinations: Animals 1535 and 1536 were both designated in the data as Diestrous at termination, not

estrous. Corrected. OK

48–Appendix W, Animal 4549, print-out dated 4/4/03: There was a correction page dated 4/30/03 that indicates the organ was supposed to be designated ‘pituitary’ and not ‘gross exam’. As well, the finding of incisors broken/ missing is supposed to be an external exam finding. I believe these corrections were made but the latest computer version might not have been used for reporting. Actually corrections were made 5/1 and 6/16 as well. Please verify that the latest version is reported. Table and Appendix re-printed. OK

49–Appendix X will need to be updated when Stillborn and Dead designations are resolved. Statuses for other pups corrected based on results of lung floatation tests. OK

50–The protocol didn’t require that culled pups be necropsied, but only observed externally for abnormalities. There is a data sheet with culled pups listed indicating ‘no gross observations’. Appendix X indicates culled pups with

‘no remarkable observations’. Were these pups necropsied? Please clarify what no remarkab le observations refers to, either internal gross or external exams? External exam only performed by the necropsy department. OK

51–Appendix X, page 14: Data for animal 3528 indicates, ‘exam not completed’. Was it completed later? What does this mean and why are no remarkable observations reported? Raw data states that the macro exam is complete as does the appendix for this dam. OK

52–Appendix Y, page 7: For animal 3029, it should indicate ‘see deviation 4’ in the spaces where there are no organ weight/ratios. Notation added to bottom of page 8 in Appendix Y. OK

53–Appendix Y, page 24: For animal 4532, there was documentation to indicate that the left ovary weight and ovaries weight were to be deleted due to tech. error in weighing. In a memo of 1/7/03 it is unclear whether these data were deleted and erroneously added back in or were these data just to be deleted? Please clarify. No changes necessary. Per memo, the tech

error was the erroneous deletion of the weights. Raw data listings were present in the data file, therefore the weights were re-input. OK

54–Appendix CC: The certificate for Jun 20 02 3B is included twice, but the certificate for Jul 01 02 2A is missing. Correct certificate added to report and duplicate certificate deleted. OK

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19 5/25/2010 

### Comments Action/Response Page

Data Issues: Individual Estrous Cycle Data: For animal 1531, for intervals 7, 8, and 9, the st age of cycle was changed on 10/28. The original data was collected on 10/14, 15, and 16, and reviewed on 10/17. How could these data be changed on

10/28? The slides for each animal for every day of smearing are retained until approval is given by study director to discard. As the slides (original raw data) are kept, the vaginal smears on days 7, 8 and 9 for animal 1531 were re-read by a technician on 10/28. Based on the final reading of the smears, a decision was made to edit the cycle to what was read

on 10/28. OK