211(b) Toxicology Research Group 

DIPE One-Generation Reproduction Toxicity Study Report 

Reviewer Checklist 

PP CS LR checklist reviewers; RG response to comment; 

1 7/27/2010 

Lab response to comments

Comments Action/Response Page 

EPA 

In general, the NHEERL reviewers concluded that the 

studies were conducted in accordance with the testing 

guidelines, once changes detailed in the API peer 

review response are made. 

No comment 

Additional comment is requested on the following 

points: 

Page 38 reports several statistically significant 

changes in reproductive organ weights (testis, 

epididymis and seminal vesicle/coagulating gland) at 

high dose exposures. The 17% increase in seminal 

vesicle/coagulating gland weight at 20,000 mg/m3 

was dismissed as being spurious, due to the absence of 

histopathological changes, and the reproductive 

NOEL was set at 20,000 mg/m3. However, since an 

increase in seminal vesicle weight is routinely used as 

a marker for androgenic activity, we respectfully 

submit that the reproductive NOEL be designated as 

10,000 mg/m3. 

THE RG does not consider the increase 

in seminal vesicle/coagulating gland 

weight to be sufficient effect to alter the 

reproductive NOAEL from 

20,000mg/m3. In addition to the absence 

of histopathologic changes, the 

parameters for sperm number, motility 

and morphology, and other reproductive 

parameters were unaffected by treatment. 

However, the sentence in Results, p. 38. 

will be revised to reflect that, rather than 

“spurious”, the weight change was not 

considered to be of definitive 

toxicological significance. 

Sentence clarified as suggested above.

P. 40 

√

The NOAEL of 

20K is also 

consistent with 

the 

interpretations 

of our external 

peer reviewers. 

Peer Reviewer – Schlesinger

The study was conducted in a scientifically sound 

manner and followed the appropriate protocols. There 

were no significant deviations from these protocols 

that would have affected the outcome of the study. 

However, there appear to be an inordinate number of 

technician errors reported. In any case, the 

conclusions as to NOELS are supported by the data. 

Nearly all of the protocol deviations 

were related to isolated errors in data 

collection. None significantly impacted 

the overall results or conclusions of the 

study. While the absolute number of 

protocol deviations may seem excessive, 

a One Generation Reproduction study is 

a considerable undertaking and involves 

a large number of animals. The fact that 

these protocol deviations were noted and 

documented reflects positively on the 

Quality Assurance practices that have 

been put in place by API, and at the 

laboratory. Nonetheless, the RG has 

brought this issue to the attention of the 

laboratory.

The following issues should be addressed in the final 

version of the report: 
211(b) Toxicology Research Group 

DIPE One-Generation Reproduction Toxicity Study Report 

Reviewer Checklist 

PP CS LR checklist reviewers; RG response to comment; 

2 7/27/2010 

Comments Action/Response Page 

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p. 28. Statistical Analysis: 2.16.1. It is noted that the 

mean values of all exposure groups were compared to 

the mean value for the control group at each time 

interval. It is not clear whether a one-way ANOVA 

was performed at each time interval so that multiple 

ANOVAs were thus used. Is there any reason why 

two way ANOVA was not used? This would have 

allowed evaluation of exposure groups at all times for 

each endpoint. This could have been followed by the 

appropriate multiple comparison test. 

The computerized statistical testing 

package employed at the laboratory is 

designed to perform one-way ANOVA 

at each time interval and is used for a 

range of multiple dose studies. The text 

will be clarified. 

Text in 2.15.1 clarified for 1-way test. 

I did not see a 

revision__same 

text but 

wording is 

same or similar 

to that in 

accepted 

reports 

Why were tests conducted at both the 1% and 5% 

level of significance. The tests and level to be used 

should be determined a priori before the study begins 

and only one such level of significance should be 

used. 

These levels of significance were preselected since they were inherent in the 

laboratory’s computer statistical package. 

Comparison at two levels of significance 

has been common practice in this 

laboratory. 

It is noted that, "Dose groups were eliminated from 

statistical analysis if their standard deviation was 0 

and/or N...in the group was less than or equal to two." 

Some indication should be noted as to how many such 

cases were encountered in the study. 

This is a standard statement for the 

statistical methods section. In this study, 

Table 1 Summary of Survival and 

Pregnancy provides examples of where 

this statement is relevant. These 

examples include data for Total females 

died/sacrificed; Nonpregnant animals, 

and No. of females cohabitated without 

evidence of pregnancy. These events are 

also observed in macro/micropathology 

incidence data. No statistical analyses 

are applied. 

p.32. Chamber Monitoring. The nominal and 

analytical concentrations are really quite close and I 

do not see a need to provide any explanation as to the 

differences. The explanation provided leads one to 

question why the calibrations of the chamber airflows 

and IR monitor were not done more carefully. 

Dr. Schesinger is correct. The sentences 

addressing reasons for possible 

differences between analytical and 

nominal values do not apply here and 

will be deleted from the Results and 

Summary sections. 

The differences were less than 3% and 

therefore considered carefully calibrated.

p.33 

reasons deleted 

√

The investigators need to explain the source of the 

particles in the exposure atmospheres. Was the 

incoming chamber air filtered appropriately? Any 

particles could act as adsorption sites for vapors in the 

exposure atmosphere. 

The mean particle concentrations were 

comparable between the control and 

treatment group chambers. The primary 

purpose of making these measurements 

in a study of this nature (high vapor 

concentrations) is to make certain that 

vapor condensation is not occurring and 

to preclude the possibility of aerosol 

formation. The results provided in the 

report confirm that gasoline + DIPE 

exposure was to vapors and not aerosols.

√
211(b) Toxicology Research Group 

DIPE One-Generation Reproduction Toxicity Study Report 

Reviewer Checklist 

PP CS LR checklist reviewers; RG response to comment; 

3 7/27/2010 

Comments Action/Response Page 

Lab response to comments

Some particles are invariably present in 

chamber atmospheres, representing 

background levels. In fact, careful 

analysis of the data in the table on page 

33 indicates that the particle 

concentrations in all of the chambers 

including controls ranged from 6-

8micrograms/cubic meter of air. For 

comparison, it has been reported in 

EPA's PM 10 Criteria document that 

annual background PM 10 levels in 

eastern cities range from 5 to 11 

micrograms /cubic meter of air. Thus, the 

RG believes that the particles in the 

chambers reflect ambient background 

values. 

p. 34. Maternal Body Weight. Effects seemed to occur 

at the mid and high exposure levels, so there does 

appear to be some consistent exposure concentrationrelated effect. 

There were no statistically significant 

changes in maternal body wt during 

gestation (absolute [Table 7] or wt gain 

[Table 8]) and no dose-responsive effects 

during lactation (Tables 9, 10) where 

values were comparable to or slightly 

higher than controls. The slightly greater 

weight gain during gestation, well within 

normal variation, is most likely due to the 

slightly larger litter sizes in the exposed 

groups. The variations in gain and loss 

patterns among the groups during 

lactation suggest that these weight 

changes are natural fluctuations in 

lactating animals and not linked 

specifically to exposure. The RG agrees 

with the study director’s assessment and 

will discuss addition of more detail on p. 

34 to justify the conclusion. 

Above discussion has been integrated 

into R/D.

p. 36 

√

Okay to me. 

p. 35. Maternal Feed Consumption. It is not clear why 

the disclaimer used here is that effects were not 

considered to be adverse while in other sections, the 

disclaimer is that effects were not in a treatmentrelated pattern. 

According to Table 13, all treated 

groups had slightly higher feed 

consumption than controls with 

statistically significant increases seen 

only in the mid dose group during 

lactation days 1-4 and 1-28. The 

observations were not considered 

“adverse responses” because modestly 

increased feed consumption (as opposed 

to decreased feed consumption) 

especially in the absence of a dose 

response pattern is not identified as a

p.36-37 

√

Higher food 

consumption 

during LD 1-4 

would be 

expected for 

larger litter sizes 

– no changes 

needed. 
211(b) Toxicology Research Group 

DIPE One-Generation Reproduction Toxicity Study Report 

Reviewer Checklist 

PP CS LR checklist reviewers; RG response to comment; 

4 7/27/2010 

Comments Action/Response Page 

Lab response to comments

potentially toxic effect. The RG will 

discuss revision of this sentence with the 

study director to clarify this conclusion.

Above discussion has been integrated 

into R/D.

p. 36. Parturition. The last sentence concerning 

statistical differences is unclear. 

The statistical differences questioned by 

the reviewer as" unclear" relate to 

“better-than-control pup survival during 

days 1-4”. 

This section has been clarified. 

p.38 

√

Okay to me. 

p. 37. Body Weight. The statement concerning litter 

size effects on statistical analysis is not clear. Table 18 

appears to show a real effect. 

From Table 16, it is clear that the 

Control group litters contained fewer 

pups by d. 4 of lactation than any 

treatment group: 11.1 in Controls, 

compared to 12.8, 13.4, and 12.5 

pups/litter in low, mid and high dose 

groups respectively. The more pups 

competing for the dam’s milk supply, 

the lower the average pup weight/litter 

resulting in the statistically significant 

lower average pup wt in the 

10,000mg/m3

 group and similar lower 

wt in the low and high dose groups 

compared to controls. When litter sizes 

were standardized on d. 4 (all litters with 

>10 pups were reduced to 10), all 

subsequent weights were comparable 

among treated groups and controls. An 

analysis of covariance could verify the 

relationship between litter size and 

average pup wt for these data. However, 

the RG agrees with the study director’s 

interpretation but will discuss including 

more detail in the explanation. 

This section reviewed again and 

considered OK as is.

Okay to me. 

p. 37. Terminal Estrous and Macroscopic Postmortem. 

Data in Table 19 seem to show an exposure 

concentration-related effect in lung, discolored foci 

Thus, this may be treatment related. 

Dr. Schlesinger is correct that the 

number of rats with discolored foci in 

the lungs was observed to increase in an 

exposure-related manner in both sexes at 

necropsy of parental animals. As 

reported in the text, no histopathological 

effects such as alveolar macrophage 

aggregates consistent with discolored 

foci were seen and no changes in lung 

wt occurred. Therefore, the increased 

frequency of these background lesions is 

likely a marker of substance exposure 

but is not considered to be of adverse 

biological significance. The RG will 

discuss revisions of the text with the 

study director to better reflect this 

assessment. 

This section reviewed again and 

p.39 

Ok to me. 
211(b) Toxicology Research Group 

DIPE One-Generation Reproduction Toxicity Study Report 

Reviewer Checklist 

PP CS LR checklist reviewers; RG response to comment; 

5 7/27/2010 

Comments Action/Response Page 

Lab response to comments

considered OK as is.

p. 38. Organ Weights. Does the lack of correlation 

with histopathology indicate no adverse effect in spite 

of potential enzyme induction induced by exposure? 

Increased liver weights (absolute and 

relative) in the absence of corresponding 

histopathology findings are often seen to 

reflect a generalized adaptive metabolic 

response to exposure in studies of a 

variety of chemicals, rather than a 

unique adverse effect of an individual 

test material. The effect plays a 

significant role in establishing the 

NOAEL (see Conclusions, p. 39). The 

text on p. 38 of the Results section 

accurately describes the events in the 

liver. No changes in text have been 

made. 

Ok to me. 

Peer Reviewer – Goldsworthy

The study was scientifically sound and was properly 

conducted. Exposures were properly conducted and 

adequate to test the potential reproductive toxicity of 

Gasoline DIPE Vapor Condensate. None of the 

protocol deviations noted would be expected to 

adversely affect study results or data integrity. 

Protocol deviations listed on page 31 did not note the 

temperature or humidity excursions outside the 

specified range (page 19). 

Excursions outside the acceptable 

temperature and humidity range have 

also been cited by a RG internal 

reviewer. 

We do not consider these temperature 

and humidity excursions to be protocol 

deviations. 

p.19 

no change to 

text 

Response from 

SD elsewhere 

is that these 

excursions 

were consistent 

across groups. 

No effect of treatment was noted on survival, clinical 

observations, body weights, feed consumption, estrus 

cycles, mating, fertility or gestation indices, or on 

other reproductive parameters up to parturition. No 

treatment differences were observed with pregnancy 

rates, duration of gestation and percent of females 

completing delivery. No differences between treated 

and control groups were noted in all parturition 

parameters, including total number of pups delivered, 

viability and lactation indices, the number of 

implantation sites per litter, the pup sex ratio, and the 

number of live pups per litter. Additionally, sperm 

count, motility, morphology and caudal epididymis 

weight were not altered by treatment. 

No comment 

There were no exposure-related differences in 

macroscopic postmortem assessments. Increased 

kidney weights were observed in male rats (parental 

generation) at the two highest exposure levels; this is 

consistent with the expected induction of hydrocarbon 

nephropathy. Microscopic evaluation was not 

performed to confirm this observation. Hydrocarbon 

nephropathy is generally not considered to be relevant 

for human risk assessment. There were statistical 

Dr. Goldworthy’s comment is correct. 

Although the Material & Methods 

section (Table 1, p. 25) stated that 

kidneys would be examined 

histologically from male rats in the 

control and high dose group, the data 

were not included in the draft report. 

The data will be added to the revised 

draft and results and discussion will be 

clarified. There were no findings of 

hydrocarbon nephropathy in the males

OK 

I recommend the 

following change 

to the text in 

Section 3.4.2: 

From: “However, 

these same 

findings were not 

seen 

microscopically 

with these test 
211(b) Toxicology Research Group 

DIPE One-Generation Reproduction Toxicity Study Report 

Reviewer Checklist 

PP CS LR checklist reviewers; RG response to comment; 

6 7/27/2010 

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differences noted in increased liver weights in male 

rats from the mid and high dose treatment groups. 

There were no exposure-related difference in body 

weights, macroscopic postmortem evaluations and 

organ weights in the lactating pups from Air Control 

animals. 

of this study.

After further review, only the kidneys of 

1049M and 4044M were examined 

because they had gross findings, as per 

protocol. Table 1 on p. 25 corrected for 

the kidneys. 

animals.” 

To: “Microscopic 

examination of 

control and high 

exposure group 

kidneys was not 

conducted in this 

study.” 

In summary, a NOAEL of 2,000mg/m3 was 

determined based on the increased liver weights 

observed in the 10,000 and 20,000 mg/m3 exposed 

parental male animals. There was no effect at any 

exposure level on reproductive performance. Thus, 

with respect to reproduction performance, a NOAEL 

of 20,000 mg/m3 was concluded. I concur that these 

data support these conclusions. 

No comment OK 

From report 

conclusions: 2K 

is the NOEL not 

a NOAEL 

[which is OK to 

me] 

211 (b) Research Group Reviewer 

General Comments 

The report should have sequential numbering from 

start to finish. 

Will be done for final report. Ok 

ok 

All of the reports should have an appendix 

summarizing historical control data for reproduction 

endpoints. Hopefully this can be included for all of the 

reports. 

Appendix DD added. Ok verified 

ok 

Specific Comments 

Page 5, 4th paragraph: Please replace "reasonably 

close" with "acceptably close," since there should be 

criteria for satisfactory comparison. 

Done although we do not have criteria for 

acceptance of this data. 

p.6 

ok 

ok 

Please consider deleting the sentence beginning, "The 

exact cause of the differences ..." since this is 

unnecessary. 

Done. p.6 

ok 

ok 

Page 19, Temperature: 

The actual temperature range high end, 31.7C, seems 

quite hot. Please explain how long this excursion 

lasted and which groups were affected. 

Excursion lasted for approx. 6.5 hours 

and affected all groups. No change made 

to report at this time.

Ok 

ok 

Page 21, Section 2.12. I, Estrous Cycling: 

Please note the study days for the start of the daily 

vaginal smears. 

Done. Ok 

ok 

Page 22, Section 2.12.4, Litter Evaluations, 

Observations: 

Please make note of the lung floatation test to 

distinguish stillborn and found dead pups, also that 

autolyzed remains found on the day of parturition 

were presumed to be stillborn. 

Done. Ok 

ok 
211(b) Toxicology Research Group 

DIPE One-Generation Reproduction Toxicity Study Report 

Reviewer Checklist 

PP CS LR checklist reviewers; RG response to comment; 

7 7/27/2010 

Comments Action/Response Page 

Lab response to comments

Page 31, Protocol Deviations, No. 6: 

Please provide the possible dispositions of the extra 

animals. Although the true disposition may not be 

known (or may simply not have been documented), it 

should still be clear that only humane options existed 

for their disposition. 

Added that “...The animals would have 

been euthanized or transferred to the inhouse colony.” 

Ok 

ok 

Page 32, Section 3.1, Chamber Monitoring, 1st 

paragraph: 

The second sentence indicates that oxygen levels were 

at least 20%. I did not find a table with the chamber 

oxygen levels; please identify. Table VI in Appendix 

A indicates that the room oxygen level was 20°/0, but 

no values exceeded that. 

Revised to “.....Chamber monitoring 

(see Table VI in Appendix A) showed 

that the chamber oxygen levels were 

20%.....”

p.33 ok 

ok 

Chamber temperatures went as high as 28C; please 

indicate when this happened, if it happened to all 

groups, and how often. 

The temperature only reached 28C on 

Exposure Day 62 for Groups 3-4 

chambers. 

No change _ok 

ok 

Last paragraph: Please consider revising "varied 

somewhat" to "varied within acceptable limits," since 

these variations were either within operating standards 

or were not. 

Done. Ok bottom of 

p33 

ok 

Page 33, 2nd paragraph: 

Please consider revising "a reasonably close" to "an 

acceptably close" to indicate that operating standards 

were met. 

Done. Ok 

ok 

Page 33, Section 3.2, Mortality: 

This section ignores the humane sacrifices that 

occurred during gestation (No. 2536, Appendix E) and 

lactation (No. 4538, Appendix F). Since these are not 

mentioned in the gestation or lactation sections of the 

results, they should be mentioned here, and 

Appendices E and F should be referenced. Also, Table 

1 does not indicate these humane sacrifices. These are 

noted in Tables 3 and 4; therefore these tables should 

also be referenced for the mortality section. 

Section revised for text and references. Looks OK 

Table also 

ok 

Also, it should be noted that No. 2536, sacrificed on 

GD 1, is presumed to have been pregnant. This is too 

early for objective confirmation of implantation. 

Agreed but leaving as presumed 

pregnant, based on implantation sites, 

does not affect other parameters other 

than fertility index.

ok 

Page 36, Section 3.2.9, Parturition and Litter Survival: 

Please rephrase the last sentence; it reads awkwardly. 

Also, it would be helpful to have comments to explain 

why Group 1 lost so many more pups in the early 

postnatal period, as well as the early survival 

historical control range. 

Revised ok 

Section 3.3.2, Pup Body Weight Data: 

Table 18 appears to include every possible interval for 

body weight gain. I don't think this makes the best 

This is limited by the TASC computer 

software and cannot be modified.

Ok 

ok 
211(b) Toxicology Research Group 

DIPE One-Generation Reproduction Toxicity Study Report 

Reviewer Checklist 

PP CS LR checklist reviewers; RG response to comment; 

8 7/27/2010 

Comments Action/Response Page 

Lab response to comments

sense. Please consider reducing the number of 

intervals. 

Page 37, Section 3.14.1, Terminal Evaluations: 

Please consider if the meaning regarding lung 

discoloration is that the findings are not treatmentrelated, or not adverse. There are greater numbers of 

discolored lungs in the exposed groups, which has 

also been observed in some of the other 211b 

reproduction studies. The lack of effects upon lung 

weight or histopathology are reasonable justifications 

for the position that the discoloration was not an 

adverse observation, but I'm not as convinced that this 

can be used to indicate that it wasn't treatment-related. 

Discussion revised to indicate treatmentrelated but non-adverse pattern of 

findings. 

Ok 

ok 

Page 38, Section 3.4.3, Sperm Evaluations: 

Please correct the reference from Appendix BB to 

Appendix AA. 

Done. Ok 

ok 

Page 39, Section 3.4.5, Pup Organ Weights: 

Table 22 has two page 1s; please handle as for other 

tables with a preface page. 

Also, in the evaluation of thymus weight, the 

variability of this parameter, as observed in the 

historical control range, might be useful for the brief 

discussion of thymus weight as unrelated to exposure. 

Table 22 will be properly paginated for 

the final report. 

HCD for pup organ weights added to the 

report in Appendix DD and the 

discussion in 3.4.5. 

Ok 

ok 

Table 15 

Please include a footnote to indicate that No. 2536, 

humanely sacrificed on GD 1, was presumed to be 

pregnant and included as such in the fertility totals. 

Tables 15 and 16 footnoted. Ok 

ok 

Table 16, p. 2: 

The Group 4 number for mean pups delivered is 

shown as 13.1 here, but is summarized as 13.0 in 

Appendix R, page 4. 

Corrected. Ok 

ok 

Appendix A 

There are two page 1s; please revise for continuous 

pagination. Also, each table begins with a Page 1. 

Appendix B is handled differently (continuous 

pagination throughout the appendix) in a preferable 

format. 

Will be properly paginated for the final 

report.

Ok 

ok 

Page 1 (second page 1), Temperature: 

Please indicate when 28C occurred and how long this 

temperature lasted. 

Page 6, section 2.3.2, Chamber sampling: 

Grammatical correction: "...to characterized at least 

..." (delete d). 

This was only seen on Exposure 62 for 

Groups 3-4. 

Corrected.

Same _no 

change 

___________ 

Ok 

ok 

Appendix O, Page 4: 

No. 4538, the humane sacrifice on LD 14, has a data 

value for feed consumption for the LD 1-28 interval. 

Data presented, but excluded from 

means/stats. 

Noted that 

excluded 

OK, but still 

weird that 
211(b) Toxicology Research Group 

DIPE One-Generation Reproduction Toxicity Study Report 

Reviewer Checklist 

PP CS LR checklist reviewers; RG response to comment; 

9 7/27/2010 

Comments Action/Response Page 

Lab response to comments

This needs to be removed. there’s a value 

for an animal 

that was dead 

during half of 

the interval. 

ok

Appendix P, page 4: 

No. 4540 appears to have a single 12-day cycle 

suggestive of pseudopregnancy. Please address. 

Footnoted and data excluded from 

means/stats. 

Ok 

ok 

211(b) Research Group QA/QC Reviewer

A. Draft Final Report and Associated Raw Data 

Page 2, Compliance Statement: 

Since it was the sponsor’s responsibility to maintain 

the method of synthesis, fabrication, or derivation of 

the test fuel, and this has not been completed, it must 

be included in the sponsor’s compliance statement. 

Corrected OK 

Page 4, Quality Assurance Statement: 

Since HLS has inspected the sperm evaluation 

subcontractor (PAI) and is responsible for ensuring 

their GLP compliant status, these facility inspections 

should be listed on the QA statement for this study. 

Added by QA to the QA Statement OK 

Page 6, Summary, last paragraph, line 4: 

Regarding the exposure-related effects on male liver 

weight, a significant increase relative to brain weight 

was noted in the high-dose group. This is discussed in 

the Results section (page 38) but is omitted from the 

summary. Should the summary be revised to include 

this information? 

Added to the Summary. OK 

Page 9, Table of Contents: 

Table 18 includes pup body weight gain data, which is 

not indicated in the table title presented in the Table of 

Contents. Should the Table of Contents be revised to 

indicate that table summarizes both mean pup body 

weight and weight gain data? 

Corrected OK 

Page 13, Section 2.3, Experimental Outline: 

Footnote “b” for this table indicates histologic 

examinations were performed on reproductive tissues 

for the randomly selected control and high-exposure 

animals. Additional protocol-required tissues 

(adrenals, lungs, pituitary and lesions) were also 

examined. It is recommended that the table be revised 

to include this information. 

Table footnote revised to include that 

‘select’ tissues were also examined. 

OK 

Page 16, Section 2.5.7, Weight at Initiation of 

Exposure: 

The protocol on page 11 allows for Study 

Director discretion in accepting the outOk 
211(b) Toxicology Research Group 

DIPE One-Generation Reproduction Toxicity Study Report 

Reviewer Checklist 

PP CS LR checklist reviewers; RG response to comment; 

10 7/27/2010 

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The weight ranges for the parental males (186 to 245 

grams) and females (130 to 179 grams) were not 

strictly within the protocol specified ranges of 150 to 

200 grams for males and 125 to 175 grams for 

females. These departures from protocol 

specifications should be addressed in the data and 

report. 

of-range body weights and we do not 

therefore consider this to be a protocol 

deviation. 

Page 18, Water Analysis: 

The report text states that analyses of facility water 

samples for chemical and microbiological 

contaminants are conducted biannually (i.e., twice 

yearly). However, the water analysis reports from 

Lancaster Laboratories that are included in Appendix 

BB of the draft report present results from samples 

collected on September 25, 2000 and March 27, 2002. 

These sampling dates do not reflect biannual analysis, 

nor do they closely bracket the dates of in-life study 

conduct (May – September, 2002). Please 

correct/clarify water analysis information as 

appropriate. 

The assays have been replaced with more 

timely reports from March and August 

2002. 

ok 

Page 23, Section 2.13.1, Parental Animals, Necropsy 

Information: 

Regarding the termination of P0 males, the report text 

states that the males were euthanized after the last day 

that the F1 litters were delivered, whereas the protocol 

(Section 13.1.1.3, page 21) states that males were to 

be euthanized proximate to the date that the last litters 

were weaned. The data indicate that males were 

terminated on September 10 and 11, 2002, which was 

actually one to two weeks prior to the weaning of the 

litters. The report text needs to be revised to 

accurately reflect the termination schedule for the 

parental males. Additionally, the termination of the 

males prior to the weaning of the last litters should be 

addressed as a departure from the protocol in the data 

and final report. 

Protocol corrected in Amendment #1 

which is included with the 2nd Draft 

report to be signed by the Sponsor. 

Report text is correct as is. P0 males 

were euthanized after the last litters were 

delivered.

Ok – 

amendment1 is 

not in draft 

report 

AmendI 

received as 

separate 

document; 

requires 

signatures 

Page 24, Section 2.13.1, Parental Animals, Tissues 

Preserved and Examined Histopathologically: 

The third sentence incorrectly states that male 

reproductive tissues were examined for all P0 control 

and high exposure animals. The pathology data reflect 

that these tissues were examined from only the 10 

randomly selected control and high dose males. The 

text should be corrected to reflect actual study 

procedures. 

Corrected. Ok 

Page 25, Section 2.13.1, Parental Animals, Tissues 

Preserved and Examined Histopathologically, Table 1: 

Table corrected. ok 
211(b) Toxicology Research Group 

DIPE One-Generation Reproduction Toxicity Study Report 

Reviewer Checklist 

PP CS LR checklist reviewers; RG response to comment; 

11 7/27/2010 

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Lab response to comments

The table incorrectly indicates that brain, kidneys, 

liver and spleen were examined microscopically from 

the Group I and IV parental animals. These tissues 

were preserved but were not microscopically 

examined. Table 1 should be revised to reflect actual 

study procedures. 

Page 26, Section 2.13.2, F1 Pups, Method of 

Euthanasia: 

The text states that after Lactation Day 4, pups were 

euthanized by exsanguination following carbon 

dioxide anesthesia. This statement is not true for the 

pups from Litter No. 4538, which were sacrificed on 

Lactation Day 14, following the humane sacrifice of 

their dam. The data indicate that these pups were 

euthanized by IP injection of sodium pentobarbital. 

The text should be revised to reflect this exception. 

Also, the method of sacrifice for this litter should be 

addressed as a departure from protocol (see sections 

13.1.2.1 and 13.1.2.3) in the data and final report. 

Section revised to state that F1 pups 

culled or sacrificed prior to weaning were 

euthanized by IP injection of sodium 

pentobarbital; weanlings were euthanized 

by exsanguination following CO2 

anesthesia. 

Protocol deviation added to data and 

report.

Ok 

Page 26, Section 2.13.2, F1 Pups, Dead and Culled 

Pups: 

The report text states that partially cannibalized pups 

were discarded after examination. According to the 

study data, two partially cannibalized pups (2538-04 

and 3534-02) were apparently saved. The report text 

should be revised to reflect this exception. Also, this 

should be addressed as a departure from protocol 

Section 13.1.2.3, which states that partially 

cannibalized pups were to be discarded. 

Text corrected to indicate cannibalized 

pups were discarded or preserved. 

Protocol deviation for 2538-4 added to 

data and report. 

Per review of saved tissues, Pup 3534-2 

was not saved. No deviation necessary.

Ok 

Page 28, Section 2.15.1, Statistical Analysis of 

Continuous Data: 

The report text and summary tables indicate that 

estrous cycle data were analyzed as continuous data. 

However, the protocol (Section 15.1, pages 26 and 27) 

does not include estrous cycle data in the list of 

continuous data to be analyzed. It appears that the 

protocol should have been amended to include the 

analysis of these data. 

This will be included in Amendment#1 to 

be provided in the 2nd draft report. 

Ok – 

amendment 1 

is not in this 

draft 

Page 30, Section 2.15.3, Incidence Data Analysis: 

Should this paragraph be revised to indicate that 

incidence data were first analyzed by Chi-square test 

followed by Fisher Exact test if the Chi square test 

was significant (as per protocol section 15.2.1)? 

This will be included in Amendment#1 to 

be provided in the 2nd draft report.

Ok, 

amendment 1 

is not in this 

draft 

Page 31, Protocol Deviations: 

Excursions from the protocol-specified animal room 

temperature and humidity ranges were noted 

throughout the course of the study. It is recommended 

We do not report animal room 

temperature and humidity ranges 

excursions as protocol deviations based 

on the protocol wording of ‘maintained 

Ok - all 
211(b) Toxicology Research Group 

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that this be included in the deviations section of the 

final report. See also items 6, 8, 11, 12, 13, 32 and 38 

of this audit report for additional protocol departures 

that may need to be incorporated into the Protocol 

Deviation section of the final report. 

to the maximum extent possible’. 

6 – see previous response (Page 16, 

Section 2.5.7, Weight at Initiation of 

Exposure) 

8 - see previous response (Page 23, 

Section 2.13.1, Parental Animals, 

Necropsy Information) 

11 - see previous response (Page 26, 

Section 2.13.2, F1 Pups, Method of 

Euthanasia) 

12 - see previous response (Page 26, 

Section 2.13.2, F1 Pups, Dead and Culled 

Pups) 

13 - see previous response (Page 28, 

Section 2.15.1, Statistical Analysis of 

Continuous Data) 

32 – No protocol deviations noted. 

(Appendix B, Analytical Report, Page 

22-23, 2,3-Dimthylbutane, all samples) 

38 – No protocol deviations noted. 

(Appendix O, Individual Maternal 

Lactation Feed Consumption)

Page 36, Parturition and Litter Survival Data: 

The results section does not address pup sex ratios. 

Should this section of the text be revised to mention 

that this parameter was also unaffected by treatment? 

Section revised to also discuss sex ratio. Ok 

Page 38, Section 3.4.3, Sperm Evaluations: The 

reference for the sperm evaluation report should be 

Appendix AA (not Appendix BB). 

Corrected. Ok 

Table 1, Summary of Survival and Pregnancy: 

The summary table indicates that one pregnant female 

in Group I and Group III died or was sacrificed 

postpartum (incidences of 1 are reported under 

“Pregnant, Died/sacrificed postpartum” and under 

“Total females died/sacrificed” for each of these 

groups). Appendix C (Individual Animal Termination 

History) and the original data reflect that all females 

in these two groups were terminally sacrificed. These 

apparent discrepancies should be corrected/clarified in 

the report. 

Table 1 corrected. 

Ok 

Table 1, Summary of Survival and Pregnancy: ok 
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The summary table indicates that no pregnant females 

in Group IV died or were sacrificed 

postpartum(incidences of 0 are reported under 

“Pregnant, Died/sacrificed postpartum" and under 

“Total females died/sacrificed”). Appendix C 

(Individual Animal Termination History) and the 

original data reflect that Dam No. 4538 was a humane 

sacrifice on Lactation Day 14. Should this be included 

in the summary data presented in Table 1? Please 

correct/clarify as appropriate. 

Corrected. 

Table 3, Within Normal Limits entry: 

These counts of normality include the females that 

were not pregnant. Should they be summarized with 

the animals that ended up delivering and completing 

the study? Please comment. 

We agree it is OK and the TASC 

program tabulates in that manner. 

Ok 

Table 4, Terminal Sacrifice entry: 

for the Controls, Group 3 and 4 entries may not be 

accurate. Group One Animal 1549 was not entered as 

a terminal sacrifice in Appendix F, so was not 

summarized here. The same is true for Animal 

Number 3534. For Group 4, I count 22 animals at 

terminal sacrifice from Appendix F. Please clarify. 

Both animals were sacrificed on Day 30, 

therefore were not presented on this table 

(range was LD 1 to 28). Table re-printed 

with range set to LD 1 to 30. 

Ok 

Table 16, Summary of Delivery and Litter Data: 

Pup death and survival data on pages 1 and 2 and pup 

sex ratio on page 3 of the summary table are reported 

through day 21 of lactation. Shouldn’t these data be 

reported for the full 28-day lactation period? 

No changes possible due to TASC 

system limitations. 

Ok 

Table 16, Summary of Delivery and Litter Data versus 

Appendix R, Individual Delivery and Litter Data, Sex 

Ratio on Day 0: 

Appendix R indicates that there were a total of 155 

male pups on Day 0, whereas Table 16 reports a total 

of 150 male pups at this interval. Is this due to the 

exclusion of 5 male pups from Litter No. 4538, which 

were terminated early due to the humane sacrifice of 

the dam? Please address/clarify as appropriate. 

No change necessary. Appendix R 

clearly states that Litter 4538 is excluded. 

Ok 

Table 17, Summary of Pup Clinical Observations 

During Lactation: 

The table title indicates that incidences are presented 

as “frequency/animals”. It appears that data are 

actually presented as “frequency/number of affected 

litters”. 

No change to table title possible due to 

TASC system limitations. 

Ok 

Table 20, Summary of Pup Necropsy Observations 

versus Appendix W, Individual Pup Necropsy 

Observations: 

Appendix W reports external findings of “domed 

head” for Pup Nos. 3531-5 and 3531-13. These 

observations do not appear to be summarized in Table 

No changes necessary. Table 20 

excludes External Exam findings as these 

are confirmation of In-life findings. 

ok 
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20. Please correct/clarify as appropriate. 

Appendix A, Inhalation Report, Table V, Miran 

Calibration: 

The Average Absorbance (Volts) for the last two 

concentrations should be 0.639 and 0.777. Are these 

typographical errors or were incorrect values used for 

the regression analysis? Please clarify. 

The data is correct and the report has 

been corrected. 

Ok as long as 

correct value is 

reported 

Appendix A, Inhalation Report, Pages 1 and 2: 

Some mention as to the duration or frequency of the 

temperature and humidity excursions should be given. 

Frequency of excursions added. Ok 

Appendix A, Inhalation Report, CMR Table, Page 11: 

For the chamber concentrations on 8/25/02, it is not 

clear where the value of 3110 mg/m3came from. The 

first concentration noted was 1410 and then 5 minutes 

later the value of 3520 was recorded. Five minutes 

after that the value was 2700.Is 3110 a time-weighted 

average? Please clarify. 

Per SOP, the D1 and the D2 samples 

were averaged and the original value was 

ignored as an unconfirmed outlier.

Ok 

Appendix B, Analytical Report, Page 5, Results and 

Discussion: 

A statement summarizing the results is needed. It 

should be indicated that no drift in the major 

component proportions was noted throughout the 

study. 

Added. Ok 

Appendix B, Analytical Report, Page 5 Results and 

Discussion: 

There should also be some discussion about the 

amount of DIPE that was seen in the vapor. For some 

of the exposures (example - Exposure 84), a different 

column was used that separated the N-hexane and 

DIPE showing around 3 Area Percent N-hexane and 

18 or 19 Area Percent DIPE. This is consistent with 

the nhexane content of the other test materials in this 

program showing between 2and 4 Area Percent Nhexane. 

Added to the results and discussion 

section. 

Ok 

Appendix B, Analytical Report, Page 15, 2,3-

Dimethylbutane, Sample 3008: 

Please review the values for this component. It 

appears that the peak may have split and the retention 

time shifted. The value appears correct on the GC 

print-out. 

Correct as is. The sample peak was reidentified on the GC print-out. Ok 

Appendix B, Analytical Report, Page 22-23, 2,3-

Dimthylbutane, all samples: 

A different column was used and it appears that the 

peak may have split here as well, but wasn’t added to 

the main peak. Please verify. 

Peak areas of split peaks were added on 

the data and the report was updated. 

Ok 

Appendix D, Animal number 1540: 

This animal was a terminal sacrifice on Day 108, not 

Animal’s death is correctly entered as 

Day 108, however, because this was not a 

ok 
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105. How could it have been sacrificed on the 

computer on Day105? The same is true for animal 

3530. Please clarify. As well, on Table 2, it is not 

entered as a Terminal Sacrifice for Day 105. It doesn’t 

show up until Day 112. Please also verify that animal 

2543 was sacrificed when appropriate and that it was 

summarized correctly. 

scheduled day for clinical observations, it 

does not appear on the report on the 

actual day of death. Summary report 

(Table 2) presents deaths occurring 

between scheduled periods at the next 

interval; individual data report (Appendix 

D) presents this death on the previous 

interval. Notations have been added to 

the preface pages. 

Appendix E, Animal number 2536: 

An observation of “decreased FV” is entered for this 

animal without a corresponding footnote explanation. 

Abbreviation removed and replaced with 

full text observation (fecal volume). 

Ok 

Appendix F, Page 7, Animal No. 3534: 

This animal was not observed at the required intervals 

and appears to have not been observed at 

termination(appendix F). Please include this as a 

protocol deviation and verify that the data has been 

reported correctly, in summaries if appropriate, or 

separately if not. 

No deviation necessary. Animal lost its 

litter and was placed on a weekly 

observation schedule. See also Animal 

No. 1549. 

Both animals were sacrificed on Day 30. 

Appendix extended to Day 30 to show 

Terminal Sacrifice. 

Ok 

Appendix N, Individual Gestation Feed Consumption: 

For Group I female1531, please recheck the reported 

food consumption value for Gestation Days0-4. 

Appendix N reports consumption of 78 grams, 

whereas the raw data appears to reflect food 

consumption of 85.6 grams (572.2 grams offered on 

Day 0 and 486.6 grams remaining on Day 4). Please 

recheck and correct/clarify as appropriate. 

No change to report. Food consumption 

is reported as grams of food consumed 

per kilogram body weight per day: 85.6 

g consumed/0.274 kg body weight/4 days 

= 78.1 g/kg/day. 

0k 

Appendix O, Individual Maternal Lactation Feed 

Consumption: 

For Lactation Days 4-7, Group I females 1535 and 

1542 are reported in the appendix shaving invalid 

weights due to the amount of feed remaining being 

greater than amount fed. The study data indicate that 

these weights were excluded because the animals 

were not able to access their water valves due to faulty 

caging. The report appendix should be corrected to 

reflect the actual reason for the exclusion of these 

food consumption values. 

Appendix corrected. Ok 

Appendix O, Individual Maternal Lactation Feed 

Consumption: 

For Lactation Days 14-21, Group IV female 4530 is 

reported in the appendix as having an invalid weight 

due to the amount of feed remaining being greater 

than amounted. The study data indicate that the 

weight was excluded because of technician weighing 

error (technician forgot to tare jar with old feed prior 

Appendix corrected. ok 
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to adding new feed). The report appendix should be 

corrected to reflect the actual reason for the exclusion 

of these food consumption values. 

Appendix R, Individual Delivery and Litter Data: 

For Female No. 4530, the abbreviation “pc” is used in 

the “Duration of Gestation” column. This abbreviation 

is not defined in the notations at the bottom of the 

table. 

Abbreviation was not necessary and was 

removed. 

ok 

Appendix S, Individual Pup Sex and Status During 

Lactation and Appendix W, Individual Pup Necropsy 

Observations: 

Pup No. 2538-4 is reported as being found dead on 

Lactation Day 1. The study data (Individual Dead Pup 

and Internal Examination Record) indicates that this 

pup died on Lactation Day 0 (August 23, 2002). An 

edit dated September 25, 2002 shows that the status 

and death day were changed from missing on day 1 

(4/1) to dead on day1 (1/1), with the reason for the 

edit being “per dead pup sheet”. It is not clear, 

however, why the day of death was changed from 

Lactation Day 0 to Lactation Day 1. Please 

correct/clarify as appropriate. This correction may 

affect associated summary tables. 

Data reviewed. It appears the day of 

death edit was made in error. Data reeditted to day of death = Day 0. 

Associated tables and appendices 

reviewed and revised as necessary. 

ok 

Appendix W, Individual Pup Necropsy Observations: 

Appendix and study data reflect that lung flotation test 

was performed for Pup No. 3534-1 at the time of its 

death. Since this pup was found dead on Lactation 

Day 1, rather than Lactation Day 0, it is not clear why 

lung flotation was performed. The protocol required 

lung flotation only for pups found dead at birth. Please 

clarify in study data and report why lung flotation was 

performed. 

Protocol deviation added to data and 

report. 

ok 

Appendix Y, Individual Pup Organ Weights and 

Organ/Body Weight Ratios: 

The weights reported in the “final body weight” 

column of this appendix are the Lactation Day 21 

body weights rather than the Lactation Day 28 final 

weights. All values need to be edited to reflect the 

Day 28 values and the organ/body weight ratios 

recalculated using the Day 28 values. The associated 

summary tables with statistics appear to report ratios 

calculated using the correct (Day 28) weights, but this 

should be rechecked to verify. 

Corrected. ok 

Appendix Z, Individual Animal Gross and 

Microscopic Observations: 

For Group I female 1544, the reported gross 

observation for adrenals indicates red discoloration. 

The raw data printout dated September 20, 2002 does 

Edit dated 20 Sep 02 added “Red” to this 

finding. No change necessary (see also 

Appendix V). 

ok 
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not specify a color. Was this observation edited to 

include the color modifier "red”? Please address in 

data and/or report as appropriate. 

B. Additional Raw Data and Protocol Comments 

Study Protocol, Section 13.1.2.3, Dead and Culled 

Pups: 

The protocol states that culled F1 pups will be 

examined externally and if unremarkable, will be 

euthanized and discarded. The study records 

(Individual Pup Necropsy Observations) appear to 

indicate that culled pups were examined, at necropsy 

as an entry of “No Remarkable Observations” is 

documented for each culled pup. Please document as 

protocol deviation or otherwise clarify as appropriate. 

Not a protocol deviation since culled 

pups were examined as per protocol 

requirement.

ok 

Analytical Data: 

No standard runs were included with this study data. 

They are probably included with the DIPE Subchronic 

Study. This needs to be clearly referenced. As well, no 

certificates of analyses were present for standards. 

Correct as is: test substance standards 

are present for all runs in this study. See 

injection #003 and #009 in each run. 

ok 

Analytical Data: 

There are system calibration data for study 6129 

(10/10/01),6130 (2/4/02) and 6131 (6/6/02) and then 

print-outs for 6131 dated 8/22/02 included with study 

4205 data. It is not clear what these calibration runs 

are for and why they are included with study 4205 

data. Are they analyzing a butane standard, and 

shouldn’t they be included with their respective data 

and referenced in this data book? Please clarify. 

System calibration data from other 

studies removed from 00-4205.

ok 

The data form entitled “Tissues Saved Required by 

Protocol ”for Group I males 1039 through 1051 does 

not include spleen in the list of tissues saved. Please 

correct the form to indicate that this protocol-required 

tissue was retained for these animals. 

Clarified in data that spleens were saved 

for 8 of these males but not for 5 of these 

animals. This has been noted in data and 

report as a protocol deviation. 

ok 

The Individual Dead Pup Internal and External 

Examination Record for Pup No. 3550-1 indicates that 

that this dead pup was examined on Lactation Day 

1(August 22, 2002) and determined to be stillborn. 

Since the dam initiated delivery on August 21 but did 

not complete until August 22, 2002, this pup is 

reported as a Day 0 stillborn, although the data still 

reflects it was examined on Day 1. It is recommended 

that a notation be added to the “Dead Pup 

Examination Record” to clarify the pup’s status. 

Data has been annotated to clarify the 

pup status.

ok 

In Book 1 of 1, the edit record for the terminal estrous 

cycle data (hand recorded data) dated November 1, 

2002 does not indicate the interval being edited. 

Please add this information to the edit record. 

These edits refer to the addition of estrus 

parameters to the necropsy portion of the 

Xybion protocol and study list so that the 

terminal smear readings could be entered.

ok 

For Gestation Body Weights, the handwritten Data was corrected 24Jun04. ok 
211(b) Toxicology Research Group 

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correction sheet for Group IV female 4549 should be 

corrected to indicate “Gestation Day 4” rather than 

“Lactation Day 4” for the data being corrected. 

For Group III male 3047, an initial day 112 body 

weight of 522.1 grams was recorded. Upon recheck, 

the weight was determined to be 542.9 grams and this 

weight was reported. According to SOP TX-02-5.2, a 

second verification weight should have been taken 

since the first verification weight differed from the 

initial weight by more than 20 grams. This should be 

addressed as an SOP deviation in the study records. 

SOP deviation memo was signed 

23June04 and added to data.

ok 

It is not clear from the data whether the carcass of Pup 

No. 1528-7 that died on Lactation Day 2 (August 23, 

2002) was saved. This pup was noted as being 

autolyzed at the time of examination and is not listed 

on the “Dead Pups Saved” form. Please clarify in data 

whether this carcass was saved or discarded. If the 

carcass was not retained, this will need to be 

documented as a protocol deviation (see Protocol 

Section 13.1.2.3 which states that dead be retained). 

Clarified in data that tissues were saved. ok