Gasoline DIPE Vapor Condensate: Embryo-Fetal Toxicity Study in Rats by Inhalation Exposure 

HLS Study No. 05-4287 Checklist April 24, 2012 

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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 gravid uterine weight 

is an indicator of maternal 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. 26 
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The methods for statistical analysis of fetal 

examination findings should 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 the 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 reviewer to repeat or 

confirm these analyses. This is 

particularly important for endpoints where 

the data suggest a treatment effect (e.g., 

in this study, 14th 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-litter 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 covariate, it provides 

limited information, and should be 

removed from the model. Once removed 

there will be one statistically significant 

result (for Sternebrae, 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) 

 
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HLS Study No. 05-4287 Checklist April 24, 2012 

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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 condensation is not occurring, 

and to preclude the possibility of aerosol 

formation. We believe that the particles in 

the chambers reflect 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 represent a biologically or 

toxicologically relevant difference. Fetal 

body weight was a consideration in the 

selection of the developmental NOEL, 

10,000 mg/m3 

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 consumption. The WG 

will ask the study director to include error 

p. 31 and 

32 
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bars if this is possible within the program 

that generates the figures. 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 for 

suitable 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/m3

 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 any 

level 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 and 
Gasoline DIPE Vapor Condensate: Embryo-Fetal Toxicity Study in Rats by Inhalation Exposure 

HLS Study No. 05-4287 Checklist April 24, 2012 

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establishes a NOAEL for embryo fetal effects 

at 20,000 mg/m3

. 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 the 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 report is an independent 

document and our conclusions will not be 

affected by the EMBSI report conclusions. 

Done and Accepted 

The incidence of 14th (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/m3

, or as stated in the text, 20,000 

mg/m3

 (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/m3/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/m3

/day based upon lowered fetal body 

weight at 20,000 mg/m3/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. 210 
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HLS Study No. 05-4287 Checklist April 24, 2012 

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include rudimentary 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 conclusions 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 the 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 report 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 Table is entitled, Mean 

Uterus Weights. The 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 

157 
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weight). Please provide 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 that 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 the final report will need 

continuous pagination. 

This will be done for final report. 

Accepted 

Among the specific comments related to 

Results, I've suggested that Appendix N 

be referenced, since it is the only location 

for statistical evaluation. 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 for 

body 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. 27 
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HLS Study No. 05-4287 Checklist April 24, 2012 

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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 reviewer 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 other developmental 

findings to support growth retardation, 

although the nonstatisically different 

increase in the incidence of rudimentary 

supernumerary ribs could be viewed as a 

suggestive variant. The smaller fetal 

weights at 20,000 could be the result of 

that group having the largest mean live 

litter size. However, litter size was a 

covariate for statistical 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 conservative 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. 27 
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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 first 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 for 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 "Soft 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, Number of Animals: 

Spelling error: "guidlines" should be 

"guidelines." 

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. 11 
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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 measurements: The value for 

MMAD for group 3 is shown as 3.662 um; 

this is shown as 3.762 um in Appendix B, 

Table II. 

Corrected. 

Accepted

p. 24 

 In this text below this 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 among 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, Feed 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: There shouldn't be a line 

Lines removed. p. 32 
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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 observation. 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 (Appendix L, p. 25, no. 

3911-11). The internal observation of 

hydrocephaly is included in Table 9; again, 

please add a footnote if the external 

observation was intentionally omitted. 

(3) no. 4907-1 had a missing lumbar 

vertebral centrum (Appendix 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, Table 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 revise "Determination 

of the exposure levels were.." to either (a) 

"Determinations 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 report (dated January 12, 

2006) was audited to assure compliance 
Gasoline DIPE Vapor Condensate: Embryo-Fetal Toxicity Study in Rats by Inhalation Exposure 

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with GLP regulations (Part 79.60) and 

that the report represents 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 

Soft 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 study 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 from 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 there 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. OK 
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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 Substance, 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. 

OK 
Gasoline DIPE Vapor Condensate: Embryo-Fetal Toxicity Study in Rats by Inhalation Exposure 

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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 pagination will be added at 

report finalization.

OK 

 Calculations page: the calculation for 

food consumption appears to be 

incorrect. It is reported as g/kg/day. 

Correct food consumption calculation 

added. 

OK 

Table 7, Live Fetuses, 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, Analytical Report, Page 7, 

DIPE STD: The Tank designation should 

Corrected in the report OK 
Gasoline DIPE Vapor Condensate: Embryo-Fetal Toxicity Study in Rats by Inhalation Exposure 

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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 the 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”. The 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 reinjected 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, Environmental 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.

OK 
Gasoline DIPE Vapor Condensate: Embryo-Fetal Toxicity Study in Rats by Inhalation Exposure 

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Appendix I, Statistical Report: It appears 

that the statistical evaluation 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 statistical 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 Findings 

The 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 “for 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 

OK 
Gasoline DIPE Vapor Condensate: Embryo-Fetal Toxicity Study in Rats by Inhalation Exposure 

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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 the group effect varied 

between males and females, then it is 

more appropriate to test 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 title of this appendix 

should read Visceral, External and 

Skeletal Observations. 

Appendices L and M reviewed. With the 

exception of Crown-rump distance, all 

findings in Appendix L are also found in 

Appendix M. Therefore, 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 originally 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 

14th rudimentary rib is Left, not bilateral. 

Corrected OK 

Appendix M, Animal 3914, fetus 15, the 

14th rudimentary rib is left. 

Corrected OK 
Gasoline DIPE Vapor Condensate: Embryo-Fetal Toxicity Study in Rats by Inhalation Exposure 

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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 the 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) malaligned”, 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 the 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) malaligned”, 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) malaligned”, 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, 7th

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 gravid 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 OK 
Gasoline DIPE Vapor Condensate: Embryo-Fetal Toxicity Study in Rats by Inhalation Exposure 

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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 present 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 

OK 
Gasoline DIPE Vapor Condensate: Embryo-Fetal Toxicity Study in Rats by Inhalation Exposure 

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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 group 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 appropriate 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. 

OK 
Gasoline DIPE Vapor Condensate: Embryo-Fetal Toxicity Study in Rats by Inhalation Exposure 

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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 added 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 the nine small cylinders 

1P1, 1L1, 1O1, 1D1,1E1, 1G1, 1K1, 1N1, 

and 1Q1 which were stored in room 813 

for use on test. 

OK