Gasoline DIPE Vapor Condensate: Embryo-Fetal Toxicity Study in Rats by Inhalation Exposure HLS Study No. 05-4287 Checklist April 24, 2012

1 

### COMMENTS Action / Response Page #

EPA

Overall, ORD reviewers found that the methods employed were in accordance with the guidance of the 211(b) test rule. In general, the API response to the independent peer reviewers’ comments adequately addresses the reviewers’ comments; however, it should be noted that the RG’s responses to comments have not been incorporated into the report. The peer review comments should be addressed and reflected in the final report. A couple issues warrant further comment: Now incorporated

In the peer review comments prepared by Dr. Goldsworthy, he noted that data shown in table 8 on gravid uterine weight are not presented on the “Results Section” of the report. ORD notes further that net body weight change minus grav id uterine weight

is an indicator of mater nal toxicity, yet this endpoint is not mentioned in the test nor is it mentioned in the statistics report (Appendix N). Table 8 shows dose-related decreases for this endpoint at all dose levels, a finding which may impact the maternal NOAEL. This maternal endpoint should be statistically analyzed and the results included in the report. Section 3.3.7. added to the report to discuss this data however Appendix M (Table 4) already included this data. We included the analysis of this additional parameter in the updated report.

Accepted p. 26Gasoline DIPE Vapor Condensate: Embryo-Fetal Toxicity Study in Rats by Inhalation Exposure HLS Study No. 05-4287 Checklist April 24, 2012

2 The methods for statistical analysis of fetal examination findings s hould be clarified. The methodology section of the statistical report (Appendix N) states: These parameters were analyzed using generalized linear models (Proc Genmod, SAS Institute 1999)

with the litter as the basis for analysis. A constant correlation among littermates was incorporated

and litter-size was included as a covariate. If the dose group effect in the model was statistically significant, each dose group was compared with t he control group using pairwise test associated with least squares means, in this case being the mean incidence adjusted

for litter size. Without additional information, it is impossible for a review er to repeat or confirm these analyses. This is particularly important for endpoints where the data suggest a treatment effect (e.g., in this study, 14 th rib). Please clarify the methods. Were generalized estimating equations (Ryan, 1992) 1 used for these analyses? What models and what link functions were used? Furthermore, please justify (or consider discarding) the

use of litter size as a covariate in these analyses. Ryan (1992) points out that the approach to characterize the litter effect has been criticized as inadequate in capturing litter-to-litte r variability. In practice, we doubt that using litter size as a covariate enhances these analyses.

1 Ryan L, 1992. The use of generalized estimating equations for risk assessment in development toxicity. Risk Analysis,

12:439-447. The original Proc Genmod SAS code for the analysis was

proc genmod data=fetaldata; class group animalno; model Ysum=group littersize/dist=p type3; repeated subject= animalno /type=cs modelse; lsmeans group/diff;

i.e. we assume a Poisson distribution with a log link function. Data was analysed using Generalized Estimating equations. These details were added to the report. Accepted

We have reviewed the inclusion of litter

size as a covariate in the statistical model and agree that, given the information

contained within the cova riate, it provides limited information, and should be
removed from the model. Once removed there will be one statistically significant result (for Sternebr ae, V Sternebra(e)malaligned, p=0.047) and one nearsignificant result (Ribs, V Rib(s) 14th Rudimentary, p=0.067). These changes were reflected in the updated report. Accepted p. 145



### Dr. Schlesinger
I have reviewed the above captioned (BG Dipe Vapor Condensate Embryo-Fetal)Gasoline DIPE Vapor Condensate: Embryo-Fetal Toxicity Study in Rats by Inhalation Exposure HLS Study No. 05-4287 Checklist April 24, 2012

3 draft report. The study was conducted in the proper manner and the results presented support the conclusions reached. There were no protocol deviations that would have adversely affected the study outcome. Some specific issues that should be addressed by the investigators are as follows:

p. 27. It is noted that some particles are invariably occurring in chamber air. However, if air incoming to the chambers is properly filtered, there should be none, especially in the Air Control chamber. Any “core” particle in the

air could become a surface onto which the vapor could condense. Appropriate filters were in place, but some particles are invariably present in chamber atmospheres, representing background air. The primary purpose of particulate measurements in a study utilizing high vapor concentrations is to make certain that vapor condensati on is not occurring, and to preclude the po ssibility of aerosol

formation. We believe that the particles in

the chambers reflec t ambient background

levels.

No further action needed. p. 27

p. 29. The investigators indicate that they do not consider the effects on fetal weight in the high exposure group to be an effect of exposures since the difference from control was very small. However, this may be the only effect of the exposure on the animals and, as such, should not be so summarily

discounted. This comment is similar to a suggestion from Dr. Goldsworthy. The lab will be

asked to review their interpretation in light of typical variability within their historical

control data as well as to consider whether the slight but statistically significant differences noted for fetal body weight, 3- 4% of control values, are truly considered not to represent an effect of the exposures, as the report is currently written, or not to repr esent a biologically or toxicologically relevant difference. Fetal body weight was a consideration in the selection of the developmental NOEL, 10,000 mg/m 3

Additional discussion added to section

3.4.1. however the conclusion did not require changing. NOAEL remains as

stated.

Accepted p. 29

Figures 1 and 2. Error bars should be shown on these figures. Figures 1 and 2 represent gestation body weights and food consum ption. The WG will ask the study director to include error p. 31 and

32Gasoline DIPE Vapor Condensate: Embryo-Fetal Toxicity Study in Rats by Inhalation Exposure HLS Study No. 05-4287 Checklist April 24, 2012

4 bars if this is possible within the program that generates the figur es. If the program is unable to do this, we will not pursue this request further, since Tables 4 and 5 provide standard deviations and statistical significance.

SD bars added.

Accepted. Error bars are shown for GD 5-

21 interval



### Dr. Goldsworthy

A well written report that is complete and comprehensive. Study conduct was appropriate; protocol deviations did not impact data interpretations or study outcomes. Test substance analysis and chamber monitoring demonstrated the appropriateness of the atmospheric generation techniques and stability of test substance. Exposure concentrations were reasonably close to target, and study exposures allowed forsuitable testing of test substance for embryofetal toxicity assessments.

Some reductions were seen in maternal body weight changes and food consumption during the exposure period. Body weight changes were statistically significant at all doses and generally exposure-dose related (most severe reductions in high does group). Similarly, red nasal discharge was observed in exposed animals at all dose levels. Given the relatively minor changes observed in the low dose exposure group for these endpoints, I concur that a NOAEL of 2,000 mg/m 3 for maternal effects is appropriate. [editorial: remove ‘marginal’ when presenting body weight gains deficit in summary, conclusion and section 3.3.3] The WG will request this deletion in the Summary, Results (section 3.3.3), and Conclusion (section 4)

Deletions have been made.

Accepted p. 25 and

27

The report states no effect of exposures at anylevel on pregnancy, including pre- or postimplantation loss, fetal weights or the

incidence of fetal abnormalities and thus The WG will provide the original study (EMBSI Study No. 171734, 2008) to the HLS study director for review andGasoline DIPE Vapor Condensate: Embryo-Fetal Toxicity Study in Rats by Inhalation Exposure HLS Study No. 05-4287 Checklist April 24, 2012

5 establishes a NOAEL for embryo fetal effects at 20,000 mg/m 3 . Fetal body weights in

females and for the litter were decreased in the high exposure group. Text (page 29; 3.4.1) noted that this statistical significant finding was interpreted to not represent an effect of exposure since other signs of effects on pregnancy outcome were not observed. However, it seems possible to have decreased weight loss without always having accompanying adverse pregnancy outcomes. I suggest to examine related studies to determine if rejection or acceptance of this finding is based on a consistent basis. consideration. In t he previous study at EMBSI, administration of the test

substance was not associated with statistically significant changes in mean fetal body weight or any statistically significant skeletal anomalies (Appendix K, Statistician’s Report).

The Testing Facility will review the EMBSI report but our repor t is an independent

document and our conclusions will not be affected by the EMBSI report conclusions.

Done and Accepted

The incidence of 14 th (supernumerary, rudimentary) ribs was noted as being increased, non-statistically significantly, in the high exposure group. Data show the finding increased in both fetal and litter analysis, and

an increased bilateral effect was also

observed. Text notes that this increased level is “closer to the upper range seen previously in a single group”. It would be useful to present the historical data of non-exposed animals for the laboratory as well as to

comment within the context of all APIsponsored gasoline studies. I would also suggest a trend-test of raw data in the current study. These suggestions for a more robust analysis would aid in understanding if the NOAEL for embryo should be established at 10,000 mg/m 3 , or as stated in the text, 20,000 mg/m 3 (and would result in modifying text, including summary and conclusions). The WG will request that appropriate

historical control information on relevant developmental endpoints be included as an appendix to the report. The WG will request that the study director review the overall 211b developmental toxicity program findings for possible consideration in the interpretation of the results of this study. The study director

was a co-author on a presentation of the results of this study and others in the 211b developmental toxicity testing program in rats (Roberts, et. al, 2009) . In this poster presentation, the study was interpreted to have a maternal NOEL of 2000 mg/m 3 /day based upon reductions in gestation body weight change and food consumption at the higher exposure levels, and a developmental NOEL of 10,000 mg/m 3 /day based upon lowered fetal body weight at 20,000 mg/m 3 /day. The higher, but not statistically significant, incidence of rudimentary lumbar ribs was noted in the poster but not defined as attributed to

exposure.

HCD for Rudimentary ribs added as Appendix P.

The historical control data from Charles River Laboratories in Appendix P does not p. 210Gasoline DIPE Vapor Condensate: Embryo-Fetal Toxicity Study in Rats by Inhalation Exposure HLS Study No. 05-4287 Checklist April 24, 2012

6 include rudimentar y or supernumerary ribs. HCD from Huntingdon is not

available. The incidence of skeletal variations from the 211b program studies (Roberts, et al., 2009) supports the laboratory conclusion s that the incidence of rudimentary ribs is not due to exposure
to G/DIPE.

Text acknowledges that this study was essentially a re-run of a previous study. If analysis were completed on the earlier study,

text should state if differences between the two studies were noted in any of the assessed endpoints. Concordance may provide the ‘robustness’ for placing various findings in the proper context. It would also be useful to know if study design and/or test parameters were changed from the original study and if so, why? The WG will provide the original study (EMBSI Study No. 171734, 2008) to the HLS study director for review and

consideration. In t he previous study at EMBSI, administration of the test

substance was not associated with statistically significant changes in mean fetal body weight or any statistically significant skeletal anomalies (Appendix K, Statistician’s Report).

The Testing Facility will review the EMBSI report but our repor t is an independent

document and our conclusions should not be affected by the EMBSI report

conclusions.

Done and Accepted

Results were not stated in the Results Section for the data shown in Table 8 on gravid uterine weight. The WG will request that gravid uterine weight be included in the text of the

Results section.

Section 3.3.7. added to the report to discuss this data however Appendix M, Table 4 already included this data.

Gravid uterine weight is now included in

Results section. There were no statistical

differences. However, Table 4 in Appendix M must be

corrected. The Tabl e is entitled, Mean Uterus Weights. T he absolute value is correctly included; however, the adjusted value is not the adjusted uterine weight, but the adjusted maternal BODY weight (terminal body weight minus gravid uterine p. 26 and

42 and

157Gasoline DIPE Vapor Condensate: Embryo-Fetal Toxicity Study in Rats by Inhalation Exposure HLS Study No. 05-4287 Checklist April 24, 2012

7 weight). Please prov ide a footnote as shown in Report Table 8, and revise this

table title.

editorial: Page 10, Introduction. Last sentence, “Because the potential...” is not complete.] The WG will request t hat the first word, “Because,” be removed from the final

sentence in the Introduction.

Deleted.

Accepted p. 8



### 211(b) Research Group Reviewer

General Comments - I'm generally very pleased with the writeup of the study and the presentation of the data. A few more footnotes (mentioned below) would be helpful, but the study was

well-done.

- A reminder that t he final report will need continuous pagination. This will be done for final report.

Accepted Among the specific co mments related to Results, I've suggested that Appendix N be referenced, since it is the only location

for statistical eval uation. It might be appropriate to reference Appendix N for every section discussing data that were evaluated by statistical methods. This was done.

Statistical Report now referenced. p. 7

- There could be alternate interpretations for the maternal and developmental
NOAELs. The lab has chosen a reasonable interpretation, with the maternal NOAEL at 2000 mg/m3 and the developmental NOAEL at 20,000 mg/m3. Overall, the absolute values are complicated a bit by the slightly smaller litter sizes in the 2000 and 10,000 mg/m3 exposure groups. The maternal NOAEL is based upon transient body weight changes at 10,000 and 20,000. For maternal data, some of the intervals forbody weight changes have a doseAgreed but none of the alternate interpretations change our interpretation therefore the conclusions are unchanged.

Accepted. Maternal net bodyweight change is now statistically significant from control at 20,000 mg/3 (not evaluated earlier). p. 27Gasoline DIPE Vapor Condensate: Embryo-Fetal Toxicity Study in Rats by Inhalation Exposure HLS Study No. 05-4287 Checklist April 24, 2012

8 responsive pattern at all dose levels. The differences from control are statistically significant at all levels for GD 5-14, and early weight changes are more likely to be

due to maternal effects than in utero effects. The low exposure group, 2000, was statistically smaller for the 2000 group for GD 8-11 (statistically significant for the 10,000 group, but not the 20,000 group), and GD 11-14 (also statistically significant at 20,000). Thus I think there is the possibility of a review er proposing that the
maternal NOAEL should be lower than 2000. None of the exposed group differences from control were statistically significant for GD 5-21, the entire exposure period, but the data have a dose-responsive pattern . There were also statistically signficant reductions in food consumption with a similar pattern at body weight change. For developmental effects, both mean fetal weight and female fetal weight were statistically significantly different from control for the 20,000 group. HLS has

considered the difference to be too small to be attributed to exposure in the absence of other developmental findings. I agree

that there are no ot her developmental findings to support growth retardation, although the nonstatisically different

increase in the inciden ce of rudimentary supernumerary ribs could be viewed as a suggestive variant. The smaller fetal weights at 20,000 coul d be the result of that group having the largest mean live litter size. However, litter size was a

covariate for statisti cal analysis of fetal weight, and fetal weight is considered to be a sensitive endpoint for growth

retardation. Noted but no change made. Fetal body weight HCD from Charles River Laboratories compared and added to discussion and Appendix P.

Accepted p. 210

A conserva tive interpretation of NOAELs and NOELs could place the NOAELs at lower values, so there may be a value to asking the lab to review their presentation and "beef up" their discussion NOAELs discussion re-reviewed and NOAELs not changed

Accepted p. 27Gasoline DIPE Vapor Condensate: Embryo-Fetal Toxicity Study in Rats by Inhalation Exposure HLS Study No. 05-4287 Checklist April 24, 2012

9 of interpretations if they keep the NOAELs where they are or choose to revise the

values. The HLS study should be viewed, I believe, as a stand-alone study. When we are at the point of considering the 211b results as a whole, we will need to be mindful of the technically acceptable findings from the firs t study (body weight change affected at 20,000, food consumption at 10,000 and 20,000, so there is consistency in the findings although latitude for interpretation; there were no statistically significant differences for fetal body weight in the original EMBSI study by the original statistical procedures,

but the same trend fo r lighter mean fetal weight was observed at 20,000) as well as findings from the literature (DIPE inhalation at > ~3100 ppm [~13,000 mg/m3] resulted in an increase in rudimentary ribs as well as some maternal effects), as well as the findings for the entire 211b program. Noted

No action needed

Specific Comments (by section & page) - Page 2, Statement of Compliance: The CFR reference on page 24 lists the citation as 79.61 rather than 79.60; presumably one of these is incorrect; please check. Also, the compliance statement seems a bit skimpy compared to others. I will leave this (okay or request revision) to Chris' judgement. Appears to be correct as is. Page 24 cites the Test Guideline in part 79.61 and the Good Laboratory Practices in part 79.60, as per protocol.

Accepted p. 2

Page 4, QA Statement: Spelling error:
"Sof Tissue" should be "Sof t Tissue." Corrected.

Accepted p. 4

Page 10, Introduction: The last sentence is incomplete (preposition only), or it is supposed to be a part of the sentence that precedes it. The first word, “Because,” was removed

from the final sentence in the Introduction.

Accepted p.8

- Page 13, 2.4.4, Num ber of Animals: Spelling error: "guidlines" should be "guid e lines." Corrected.

Accepted p.11

Page 14, 2.5.2, Supplier: The company

name needs to be corrected to our name Corrected to “ChevronTexaco Research and Technology Company” p. 11Gasoline DIPE Vapor Condensate: Embryo-Fetal Toxicity Study in Rats by Inhalation Exposure HLS Study No. 05-4287 Checklist April 24, 2012

10 as of the test substance receipt dates. I
believe this should be ChevronTexaco Research and Technology Company, but please check the containers and/or the accompanying documentation to be sure. Accepted

Page 15, 2.5.10, Dispensing: Same comment regarding the correct name for the test substance supplier. Corrected.

Accepted p. 12

- Page 27, 3.2, table for particle size

distribution measurement s: The value for MMAD for group 3 is shown as 3.662 um;

this is shown as 3. 7 62 um in Appendix B,

Table II. Corrected.

Accepted p. 24

In this text below th is table, please revise "reasonably close comparison" to "acceptably close comparison," as HLS has criteria for considering analytical variation to be within satisfactory limits. Corrected.

Accepted p. 24

- Page 28, 3.3.2, Clinical Observations:

The mass observed five times for one animal in group 4 should be noted. Details added to the report.

Accepted p. 25

Page 28, 3.3.3, Body Weights: Please include Appendix N am ong the referenced data sources. Appendix N is the only location that notes statistical significance. Please also see the general comment

above re: effect levels. Reference added.

Accepted (now Appendix M) p. 25

- Page 28, 3.3.4, F eed Consumption: Please include Appendix N among the

referenced data sources. Reference added.

Accepted (now Appendix M) p. 25

Page 29, 3.4.1, Fetal Body Weights: Please include Appendix N among the

referenced data sources. Please see the general comment above re: statistical differences for the 20,000 group. I'm not

sure what more could be stated as background substantiation for the interpretation; perhaps something about typical fetal variation from historical data Reference added to Appendix M.

Fetal body weight HCD from Charles River compared and added to discussion and Appendix P.

Accepted p. 26

- Page 31, References: Typographical error: pagination shows this as "page 1 Consecutive page numbers will be added at report finalization.

Accepted - Figure 2, Mean Gestation Feed Consumption: Ther e shouldn't be a line Lines removed. p. 32Gasoline DIPE Vapor Condensate: Embryo-Fetal Toxicity Study in Rats by Inhalation Exposure HLS Study No. 05-4287 Checklist April 24, 2012

11 connecting the data points for feed consumption between GD 20-21 and GD

5-21. I realize that this may be a program

issue and difficult to resolve. Accepted

Table 9, Summary of Fetal Observations: Please provide a footnote that malformations are written in uppercase and variations are written in both upper

and lower case. This is mentioned elsewhere, but not on the summary table. Notation added

Accepted p. 43

Findings that appear to be missing:

(1) Filamentous tail (on the same fetus as anal atresia, no. 1905- 1). The skeletal findings for this fetus, absent sacral and caudal vertebrae, are in Table 9, but not

the external observati on. If the external observation is left out intentionally because the internal finding is shown, please include a footnote to this effect.

(2) The external observation of Domed cranium is missing (Append ix L, p. 25, no. 3911-11). The internal observation of hydrocephaly is inclu ded in Table 9; again, please add a footnote if the external

observation was intent ionally omitted.

(3) no. 4907-1 had a missing lumbar vertebral centrum (Ap pendix M, p. 10); this isn't mentioned in Table 9, although other

skeletal observations for this fetus are shown on the first page of Table 9. (1) “and filamentous tail” is an additional comment to the finding “Anal Atresia”. Comments are not typically included on summary tables. Filamentous tail finding was in a control animal. Skeletal findings sufficient here. Accepted.

(2) Footnote added. Accepted

(3) Missing centrum = acentric. No changes necessary. Accepted p. 43

Appendix A, page 8, Tabl e III, footnote a: Please change "co-elutes" to "co-eluted," since this is past-tense. Corrected in the report.

Accepted p. 53

Appendix B, page 5, 2.3.2, Chamber Sampling: Please re vise "Determination of the exposure levels were.." to either (a)

"Determination s of the exposure levels were..." or (b) "Determination of the exposure level was ..." Revised to "Determinations of the exposure levels were..."

Accepted p. 67



### 211(b) QA/QC Reviewer
The draft final repor t (dated January 12, 2006) was audited to assure complianceGasoline DIPE Vapor Condensate: Embryo-Fetal Toxicity Study in Rats by Inhalation Exposure HLS Study No. 05-4287 Checklist April 24, 2012

12 with GLP regulations (Part 79.60) and that the report r epresents the data collected. One hundred percent of the report text was verified and at least 20 percent of the individual and tabular data

were verified. Page 4, QA Statement: Typo in Fetal
Sof t Tissue entry. Also, it is not clear if the Statistical Report was reviewed by HLS. This should be added, as should a review of the PAI work, either by the PAI
QA or the HLS QA. Since HLS is ultimately responsible for the GLP compliance of this subcontractor, at a minimum an entry of HLS’s review of PAI

should be included. Typo corrected and review of PAI on 13July06 added to QA Statement Ok, but

there

needs to

be some

indication
in the QA
Statement
that QA

has

reviewed this report

since

2006. Page 5, Summary, paragraph 2: The objective of the st udy presented in the protocol does not indicate any elucidation

of certain fetal observations from the previous study. It merely indicates that this study will satisfy the regulatory requirement. That should probably be stated as it is on Page 10 in the

Introduction. Summary updated to include similar text

as in the Introduction. OK

Page 5, Summary, paragraph 5: The concentrations for the last two groups are not correct. They should read 10,072+546 and 19,776+ 961 mg/m3. Summary corrected. OK

Page 5, Summary, paragraph 6: The maternal weight gain noted at GD 14 is the weight gain fr om GD11-14. Summary corrected. OK

Page 6, Summary, first paragraph: It is indicated that body weight and food consumption changes were negligible at 2000 mg/m3. In 3.3.3, page 28, body weights differences were indicated as marginal, and transient.... Section 3.3.4

indicates that ther e is no notable food consumption change at this level. The Conclusion also indicates a transient, marginal reduction in both of these parameters. Please make all of these All 3 sections revised to be consistent. OKGasoline DIPE Vapor Condensate: Embryo-Fetal Toxicity Study in Rats by Inhalation Exposure HLS Study No. 05-4287 Checklist April 24, 2012

13 findings match. (Also the word ‘negligible’ appears erroneously in the sentence in the conclusion on page 30) Page 10, Introduction, last sentence: This does not appear to be a sentence. Is something missing? See previous comment re page 10. OK

Page 14, Test Substance, Section 2.5.6:

In the data there is documentation (raw data) that indicates a date received of August 2003. It is in error and should be corrected. Please verify. Data corrected OK

Page 14, Test Substanc e, Section 2.5.8. Was the purity analysis done by the sponsor performed under GLPs? This should probably be indicated and included in the GLP compliance

statement if not. Section 2.5.8. clarified to non-GLP sponsor assay and added to the GLP

statement. OK

Page 22, Fetal Skeletal Evaluations: It is clear that PAI performed the skeletal evaluations, but it is not clear how these data were reported/recorded. The skeletal findings are reported in similar

format to the visceral and external

observations. Did PAI enter data in a TASC format and transfer it to HLS, or did
HLS enter data from hand-recorded data? This should have been part of the QA

review at one or both of the laboratories. Also, is there any peer review of the

skeletal evaluations as there are in the

soft tissue evaluations? Did PAI do a certain percentage peer review? No specific QA review was necessary. PAI was given remote access to the HLS TASC system, therefore, the data was directly captured. PAI did not do a peer

review of the skeletal evaluations – it was not required in the protocol or in their
SOPs. OK

Page 27, MMAD, Group 3 value: Please verify these values. They appear to be

3.762+2.7. Corrected to 3.762+2.6 to agree with the Appendix A. OK

Page 28, Section 3.3.3, the reduction in body weight gain noted at GD 14 is the
value from GD 11-14. Corrected OK

Page 29, Section 3.3.6, Uterine Implantation Data: Normal values for this strain of rat should be included if they are being referenced. Edited statement removing need for HCD. OK

Page 29, Fetal Body Weights: The first

sentence indicates that there was no effect of the exposures of fetal weight at Discussion expanded to clarify this section as suggested. OKGasoline DIPE Vapor Condensate: Embryo-Fetal Toxicity Study in Rats by Inhalation Exposure HLS Study No. 05-4287 Checklist April 24, 2012

14 GD21. It then goes on to indicate that the marginal difference (although stat. sig.)

was not considered to represent an effect of the exposures. The statistical report (App. I) seems to suggest otherwise, reporting that females as well as

combined sexes showed statistical significance when compared to control. Please clarify. Page 29, Fetal Observations, third paragraph, second sentence: The most

common group incidence is a better way of expressing this. The laboratory’s historical control report should be referenced here if it is going to be used to compare to these data. Revised sentence.

Reference to Appendix P added. OK

Page 30, Conclusions, first sentence: There is the word ‘negligible’ in this sentence erroneously. Section revised. OK

Page 30, Conclusions: If it is decided that the objective of this study was to compare the previous study to this one, a comparison should be done. Body weight, body weight gain, and food consumption findings appear to track relatively well, while soft tissue observations were not repeated. This is a stand-alone report and will not discuss a prior report from another lab. OK

After page 30, there appear several page

1’s. Consecutive paginatio n will be added at report finalization. OK

Calculations page: the calculation for food consumption appears to be

incorrect. It is r eported as g/kg/day. Correct food consumption calculation

added. OK

Table 7, Live Fetuse s, Group 1: Please verify this value. I found 146, not 145. Appears to be correct as is. Table 7 shows 146 male fetuses in Group 1. OK

Table 8, Footnotes and reported parameters should match Appendix J. Table corrected. OK

Table 9: It appears that there are some findings not entered for some animals. For Number 1905, there are findings that are not entered on the summary for group 1. For number 4907, there appear to be more findings than are listed on the summary for group 4. It appears that all findings are present. For Animals 1905 and 4907, extended description(s) of the findings presented in Appendix M were summarized for Table 9. OK

Appendix A, Analytic al Report, Page 7, DIPE STD: The Tank designation should Corrected in the report OKGasoline DIPE Vapor Condensate: Embryo-Fetal Toxicity Study in Rats by Inhalation Exposure HLS Study No. 05-4287 Checklist April 24, 2012

15 be 5, not 3, I believe. Appendix A, Analytical Report, Table 1: The TM STD from Study 05-4287 represent which data? It is not where

these data have come from for comparison to the earlier study data. +

1. The TM STD in Table 1 is from standard injections made on 30March05 for DIPE TM stability. See Footnote ‘b’ in Table 1. OK

Appendix A, Analytical Report: Amy Farside is listed as the analyst and report writer, yet was not t he operator on any of the assays. The analyst who did work on the study is no longer employed by HLS. The laboratory manager will sign the report. OK

Appendix A, Analytical Report/data: On 8/3/05 for the first Tank #4 run, at 12:55 there is a note to “omit, system not conditioned enough”. T he next run that appears in the data is for Sample 201 at

5:04. Where are all of the additional conditioning runs? Aren’t they normally printed and maintained? The first peak of isobutene in the first injection from Tank#4 appeared to be lower than expected which is usually due to system not being conditioned enough.

This standard was rein jected at the end of the run for confirmation. The reinjection should be reported according to our SOPs. OK

Appendix A, Tables III, IV A, B, and C:
The DIPE Std. and TM Std. source should probably be given (Tank #5, 3 or 4). Corrected and see footnotes in the tables in the report. OK

Appendix A, Figure 3: I could not find the chromatogram used for this example. The chromatogram is in the data at the end of the run in sequence 003. OK

Appendix B, Environm ental Conditions: The temperature range given should

indicate the extent and duration of the ‘out of range’ values. Added. OK

Appendix B: Room Air particle sizes should also be reported, both pretest and during testing. This has not been done for any other 211(b) reports and suggest not to be done for this report for consistency. OK

Appendix I, Statistical Report, Page 2, Compliance Statement: The appropriate
GLP reference is the one that refers to the 211b Program, US EPA 79.60. The

last sentence should also indicate “There

were no deviations from the

aforementioned standards in this statistical study or report” if this is the case. If there were deviations they

should be mentioned. Current applicable compliance statement

has been added. OKGasoline DIPE Vapor Condensate: Embryo-Fetal Toxicity Study in Rats by Inhalation Exposure HLS Study No. 05-4287 Checklist April 24, 2012

16 Appendix I, Statistica l Report: It appears

that the statistical ev aluation of the fetal body weights and the discreet findings (maternal and fetal) were not carried out according to methods indicated in the protocol. Were the tests indicated in the protocol performed in addition to those

indicated in the statis tical report? Please clarify and indicated deviations where

necessary Fetal bodyweight We believe the analysis described in the protocol and that applied to the data are the same, however the protocol wording

could have been clearer.

The protocol mentions using a mixed

model but does not describe which

random effect will be included in the model to make it a mixed model – the only candidate is litter (maternal female) but perhaps this should have been stated.

In the protocol it is stated “”the analysis uses litter as the basis of the analysis”. This is simply to imply that group effects are tested against the litter variability and not the pup variability (litter is the experimental unit). This is true if litter (the maternal female animal) is fitted as a

random effect in the mixed model.

Discrete FindingsThe protocol does not describe the nonparametric methods that were applied to some of the findings. This was performed due to the low incidence of findings that resulted in non-convergence in the GEE analysis.

This is detailed in the stats report. OK

Appendix I, Statistical Report: The units

of measure should be included where group dose levels are mentioned (mg/m3). The report was amended to reflect this. OK

Appendix I, Statistical Report: Page 7, Fetal Body Weight, second sentence: It

indicates that “f or the pooled sex analysis there was a strong sex difference”. If it is a pooled analysis, why bring up a sex difference? Wouldn’t that be discussed separately? The purpose of the pooled analysis was to assess the effect of dose group overall. As expected there was a significant difference between males and females, so sex should be included in the pooled analysis

to account for the variability between males and females. For the overall group test (averaged over males and females) to be valid, the group effect should be similar

in both sexes. To do this we include a OKGasoline DIPE Vapor Condensate: Embryo-Fetal Toxicity Study in Rats by Inhalation Exposure HLS Study No. 05-4287 Checklist April 24, 2012

17 sex*group interaction in the statistical model. As long as this is not significant (p=0.715 in this case) then pooling the

Dunnett’s test over males and females is sensible. If the interaction was significant,

i.e. the size of t he group effect varied between males and females, then it is more appropriate to te st the group effect separately for males and females. These tests (separately for each sex) are slightly less powerful when compared to the overall dose group test due to the reduced sample size.

The significant sex effect result was mentioned in the report for completeness. If it (and the group*sex interaction) were not significant then it can be argued that both terms should be dropped from the statistical model before making the overall group comparisons. This was not the case

here as the sex effect was significant. Appendix M, The ti tle of this appendix should read Visceral, External and
Skeletal Observations. Appendices L and M revi ewed. With the exception of Crown-rump distance, all findings in Appendix L are also found in Appendix M. Theref ore, Crown-rump distance was added to Appendix M, and

title was corrected to include “External”. Appendix L was removed and Appendix M to R were redesignated as Appendix L to
Q. OK

Appendix M, Animal 1904, The renal papilla finding is orig inally recorded as
Grade 1. Added. OK

Appendix M, Animal 4901. Skeletal data for this animal could not be found, yet there is a finding reported. Please verify

that this animal’s data are located in the

data notebook. Original raw data print appears to have been misplaced. A copy was re-printed

and added to the data book. OK

Appendix M, Animal 2910, fetus 8, the

14 th rudimentary rib is Left, not bilateral. Corrected OK

Appendix M, Animal 3914, fetus 15, the

14 th rudimentary rib is left. Corrected OKGasoline DIPE Vapor Condensate: Embryo-Fetal Toxicity Study in Rats by Inhalation Exposure HLS Study No. 05-4287 Checklist April 24, 2012

18 Appendix M, Animal 3921, fetus 6 and 8, there appear to be two different
corrections. One takes the numbers 5

and 6 out to not mention numbers and

one seems to add t he numbers 5 and 6, sternebrae malaligned? Please verify that what is reported is intended. Appears to be reported correctly. We see no edits adding 5 and 6 back in. Edit request says to change the finding from “Sternebra(e) 5 and/or 6 unossified” to “Sternebra(e) malalign ed”, but to keep the grade and the comment for each fetus. OK

Appendix M, Animal 3924, fetus 4, should the sternebrae malaligned be indicated for #4, 5 and 6 as noted in the edit, or with no number designation? Please verify that t he correct designation is reported. Appears to be reported correctly. We see no edits adding 5 and 6 back in. Edit request says to change the finding from “Sternebra(e) 5 and/or 6 unossified” to “Sternebra(e) malalign ed”, but to keep the grade and the comment for each fetus. OK

Appendix M, Animal 3922, fetus 2, should sternebrae malaligned 5 and 6 be added, or should no numbers be reported? Appears to be reported correctly. We see no edits adding 5 and 6 back in. Edit request says to change the finding from “Sternebra(e) 5 and/or 6 unossified” to “Sternebra(e) malalign ed”, but to keep the grade and the comment for each fetus. OK

Appendix M, Animal 4909, fetus 6, sternebra malaligned, grade should be slight as noted in data. Added. OK

Appendix M, Animal 4921, fetus 1, 7 th
cervical rib present: add ‘right’ Corrected. OK

Appendix M, animal 4924, fetus 1, renal A papilla not fully developed, is a grade

1. Added. OK

Appendix J, Animal number 4924
delivered 5 fetuses before it was sacrificed. These fetal weights are

included with the ones that were not delivered. So the gr avid uterine weight

includes both kids of fetuses. Is it appropriate to include this animal and the fetuses in with the rest of the group?
Please comment. Should the data be reported and footnoted? Data excluded and footnoted. OK

The SOP for the soft tissue examination (second read) indicates that 25% of each litter will be re-read. It does not appear that this many fetuses were reviewed (all litters were not represented). Please clarify. SOP deviation has been generated for lack of second reads. All majors appear to

have been reviewed and all litters were

examined. OK

SOP was updated to clarify that the read is OKGasoline DIPE Vapor Condensate: Embryo-Fetal Toxicity Study in Rats by Inhalation Exposure HLS Study No. 05-4287 Checklist April 24, 2012

19 The SOP for soft tissue examination

needs to be clarified. It needs to

indicate that the second review is a blind read if that is the case (there was some disagreement on this issue). The SOP quoted below seems to indicate that the peer or second review is not an independent review as they may have the original findings present.

It is unclear how the decision on what is to be reported is made and
how it is documented. The SOP quoted below did not appear to be

followed as the documentation described was not present . The following differences were noted between the original read of soft tissue and the “peer review” read.

There was no documentation to indicate that the peer reviewer and the original reviewer agreed on what was actually reported.

 1921, fetus 12: testis displaced noted by peer reviewer while testis displaced as well as undescended thymus noted by original reviewer (and reported in draft).

 3911, fetus 8: The hypoplastic lung lobe noted by the peer

reviewer but not seen on the original read was not reported. The undescended thymic tissue

as well as the left umbilical artery were seen by the original reviewer and reported but were not confirmed by the peer

reviewer.

 3911, fetus 11: Hypoplastic thymus was noted by the original reviewer and reported but was not confirmed by the peer reviewer.

 4924, fetus 1: The original

reviewer noted Ureter distended and it was reported but was not not blind and also that 25% of the study

not the litter will be reviewed.

This has been addressed in the SOP deviation generated for the lack of peer

review.

Ok as is – thymus not necessarily viewable during second read.

Ok as is – finding pres ent in original data.

Thymus – as above. Umbilical artey – see SOP deviation on lack of discrepancy check during second

reads.

Thymus – as above.

Ureter – see SOP deviation OK

OK

OK

OK

OK

OKGasoline DIPE Vapor Condensate: Embryo-Fetal Toxicity Study in Rats by Inhalation Exposure HLS Study No. 05-4287 Checklist April 24, 2012

20 confirmed by the peer reviewer.

The SOP presently indicates – A minimum of 25% of the fetuses within each litter will be subjected to a second read or peer review using Wilson’s second read recording sheets. The findings already recorded
in TASC will be checked and verified. Any resulting changes to observations in TASC, mutually agreed by first examiner and peer reviewer will be

written on the Wilson’s second read sheet, then entered on the CRDSA sheet as requiring an edit. Any resultant changes to observations recorded manually (on the Wilson’s Supplemental Recording sheet), mutually agreed by first examiner and peer reviewer will be written on the

Wilson’s second read than amended on the original sheet or entered in to TASC as raw data, fully signed and dated by the reviewer.

Data Issues In the assignment to group records, it appears that Animal number 3919 is included in the gr oup of animals with
GD 5 of 8/9/05. The clinical observations of 8/8/05 would suggest

that it is in the previous group with the
GD 5 of 8/8/05. Please clarify, and verify that the animal is in the appropriate GD 5 group and that it was sacrificed on the appropriate day. Clinical observations were collected daily from day after receipt for all gestation days; TASC allowed observations for different gestation days to be collected together. Per sort, body weight data

intervals & terminal sacrifice data for animal #3919 (pretest #90) is in Set D and was sacrificed on appropria te day of 25-Aug-05. OK

The tissue transfer sheet for the skeletal specimens going to PAI

indicates an animal number 4903 that is crossed out and changed to 4908. In the report, however, there is no
skeletal evaluation for 4908. There is

a skeletal evaluation of the fetuses from 4903 (7), even though there was
no litter from 4903 sent to PAI. Since 4908 was not pregnant, it seems unlikely that these are fetuses from This was altered by the receiving department (PAI). It was then re-corrected by HLS upon re-receipt. The initial carbon copy retained by HLS shows correct

numbers were sent out. OKGasoline DIPE Vapor Condensate: Embryo-Fetal Toxicity Study in Rats by Inhalation Exposure HLS Study No. 05-4287 Checklist April 24, 2012

21 that dam, and they are probably from 4903. Please clarify in the data why the inventory was changed and how

data could have been recorded for

dam number that was never received.

The dispensing log for the test material needs to be updated to include the
return of containers 1 and 2. And 3 when it happens. A dispensing Log for Tank no. 4 is needed with this study data as chamber trials were performed using Tank no. 4. Return of containers is not documented on dispensing log. See Pharmacy records for

details on return of containers 1 - 3.

Exact copies of Tank #4 Dispensing Log from study 00-4205 ad ded to Study File. OK

Dispensing log - There is no

documentation to indicate where the test material samples were dispensed
from. Container number is listed as ‘cylinder 9’. Please clarify. Note added to Dispensing Log that “9 cylinder” refers to t he nine small cylinders 1P1, 1L1, 1O1, 1D1,1E 1, 1G1, 1K1, 1N1,
and 1Q1 which were stored in room 813

for use on test. OK