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Response to Independent Peer Reviewers Comments: 13-week Whole Body Inhalation 

Study of Baseline Gasoline + Diisopropyl Ether Vapor Condensate and Satellite Studies

In the 211(b) testing program, the draft reports of all 13-week inhalation and satellite studies are 

reviewed by two independent peer reviewers: Dr. Thomas Goldsworthy, Consultant, and Dr. 

Richard Schlesinger, Pace University, New York, NY. Those peer reviewers have now provided 

their comments on the draft reports for studies with baseline gasoline + DIPE vapor condensate 

(BG + DIPE VC). The studies include a 13-week inhalation study including neurotoxicity and 4 

satellite reports for genetic toxicology- micronucleus and sister chromatid exchange (SCE), 

immunotoxicity and glial fibrillary acidic protein (GFAP) analysis. All studies were performed with 

animals exposed to BG + DIPE VC under the same chamber conditions. For the genetic 

toxicology and immunotoxicity studies, animals were exposed for 4 weeks; for GFAP analysis, 

animals were exposed for all 13 weeks and samples were collected at terminal sacrifice of the 

subchronic study. This draft report set includes the revised statistics for sister chromatid exchange 

and neurobehavioral endpoints recommended by EPA.

The reviewers concur that, overall, the studies were scientifically sound, protocols were 

appropriately designed and accurately followed, and results and conclusions reflect the data 

presented. None of the reviewers‟ comments or recommendations invalidates the studies. The 

211(b) Working Group (WG) responses to specific comments are organized by study and reviewer. 

Where comments were similar from both reviewers, the response is addressed jointly. 

Recommendations accepted by the performing laboratory‟s study directors will appear in final 

reports of all studies.

13-week Subchronic Inhalation Study [Huntingdon, USA]

General Comments:

Dr. Goldsworthy suggested that the titles of associated study reports be specified on pp. 34, 35, 

and 36.

Response: Rather than including the extended study titles, the WG will direct the study director 

to refer to these studies as Sister chromatid exchange report [BioReliance], Micronucleus report 

[Huntingdon, UK] and Immunotoxicology report [Immunotox, Inc].

Dr. Schlesinger recommended that consistent criteria for data evaluation be employed, especially 

levels of significance for all aspects of the study, including satellite reports.

Response: Levels of significance are identified variously as p=0.05 and p=0.01 in different 

studies because satellite studies were performed in different contract laboratories with different 

standard protocols for levels of significance which are not adjusted from client to client. To 

accommodate client needs, some contract laboratories [e.g.Huntingdon, US] provide both 0.05 

and 0.01 levels of significance for the same data set and both values are often identified in 

standardized statistical packages for computers.

Summary [p.6]

Dr Schlesinger noted that the correspondence between the measured and nominal 

concentrations was quite good and that the sentence describing reasons for occurrences when 

the correspondence was not good does not apply and should be deleted.

Response: The WG agrees with this comment. The inappropriate sentence [1st paragraph, line 

7] will be deleted.

Dr. Goldsworthy suggested that the information on motor activity [2nd paragraph, p.6] be 

expanded in the summary to correspond to the body of the report. Text should indicate that 
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statistically significant differences observed in low and medium exposure groups were not 

considered associated with BG+DIPE VC treatment because changes were not consistent 

among dose levels.

Response: The observed changes in motor activity and the reasons for not considering them 

treatment-related are well described in the report. There was no linear relationship between 

exposure levels or any consistent change in activity related to exposure. The purpose of the 

summary is to highlight conclusions without extensive detail. The WG does not consider it 

necessary to expand the Summary with additional motor activity detail.

Section 2.11.5 [p. 23] Administration of Test Substances

Dr Schlesinger pointed out that the group numbers are inconsistent as I, II, III, IV in the text and 

then 1, 2, 3, 4 in the Tables.

Response: These variations result from use of Arabic numerals in the computerized data 

gathering system, which can‟t be altered and the routine use of Roman numerals in the text. 

The study director will be requested to revise the text numbers to match the tables. 

Section 2.20 [pp42-43] Statistical Analysis

Dr Schlesinger:

Inquired why each time interval was analyzed independently and requested an explanation of 

why a 2-way ANOVA with time as a parameter was not used instead. He also requested 

clarification on which post-hoc tests were used for which data sets.

Response: A single computerized statistical testing package was employed for all parameters 

listed on p.43- 44 of report. In this package, analyses by individual sex or sexes combined 

were programmed to be performed separately by one-way ANOVA, rather than analyzed 

together by two-way ANOVA. Terminal and recovery data were also analyzed separately. 

After discussion with the study director, it was decided that additional justification for the 

statistical method employed routinely for repeat-dose studies throughout this testing facility was 

probably not needed. Revisions of statistical methods for neurobehavioral parameters were 

requested by EPA and are outlined in this section.

Thought that the use of both 5% and 1% significance levels for some endpoints did not have 

adequate justification and that only one level should be used. 

Response: Contract laboratories provide both levels of significance to satisfy needs of a range of 

clients and both values are often identified in standardized statistical packages for computers.

Inquired about the meaning of the statement under Motor Activity and FOB data that “separate 

analyses by sex may have been done to explain the nature of the effect”.

Response: Additional analysis of neurobehavioral endpoints was requested by EPA for 211(b) 

subchronic studies. That work was performed by ExxonMobil Biomedical Sciences (M. Nicolich 

and G. Bukhbinder) and used to write the draft report. The WG will instruct the study director to 

work with the contract statisticians to further clarify the study report. 

 

Section 3.1 [p. 48] Chamber Monitoring.

Dr Schlesinger requested an explanation of why “some particles are invariably occurring in 

chamber air”. He assumed that if chambers were properly filtered there should be no particles.

Response: The exposure chambers draw in air room the surrounding exposure room to dilute 

the evaporated test substance. The surrounding room air is conditioned for temperature and 

relative humidity but is not filtered to any substantial extent. Therefore, the chamber air for all 

test groups is reflective of the room air during the conduct of a gas or vapor exposure. This 

laboratory typically finds room air to contain background particles on the order of 10-3 mg/m3
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which was the range for this study. The WG will ask the study director to clarify this issue in 

the report.

Section 3.4 [p. 50] Body Weight

Dr. Schlesinger suggested that the weight gain seen in the 10000mg/m3group females might 

indicate that “something is different about these animals.” 

Response: The higher body weights in the 10000mg/m3female rats and the statistically 

significant (p<0.05) increase in overall weight gain (wk 0-12) originated back at the beginning of 

the study. Although all groups were matched for body weight at 1 week prior to study initiation, 

the 10000mg/m3females already weighed 3 grams more by week 0 the start of the study. It 

would appear that the rats in this group grew faster than other groups including the controls 

and that the weight gain was unlikely to be related to exposure. Moreover, as stated in the 

report,” increased weight gain is not usually considered an adverse response to treatment”.

Section 3.6 [p. 50] Feed Consumption

Dr. Schlesinger noted that both male and female high dose rats showed increases feed 

consumption beginning at week 14 and requested that this observation be included in the text.

Response: Weeks 14-17 constitute the untreated recovery period for this study. Although slight 

increased feed consumption occurred [Males 2-3 g; Females 2-4g, 7g at wk16] compared to 

controls, increases were not statistically significant and were not of sufficient magnitude to be 

considered reportable in the text. The WG does not consider it necessary to amend the report.

Section 3.7.1 [p.50] Motor Activity

Both peer reviewers had comments on this section.

Dr. Goldsworthy recommended that the reasons for discounting the statistical significance in 

motor activity (MA) in the low and mid dose groups be added to the Summary. The WG did not 

consider it necessary to expand the Summary with this information (see Response to Summary 

on p.1)

Dr. Schlesinger :

Is the response difference between low and mid groups found in the Pretest MA or in the data

obtained during exposure?

Response: MA changes occurred in the exposure data. As indicated in Table 8, which includes 

the analyses of data report from ExxonMobil Biomedical Sciences (M. Nicolich and G.

Bukhbinder), the pretest response is used as a covariate in the mixed model analysis of 

covariance and is not analyzed independently.

Requested clarification of the basis for the statement that “there is an increase in activity in the 

females at mid-exposure during the 4th week”.

Response: This comment appears inaccurate. The statement in the report says “data plots do 

not suggest a consistent exposure related response, but show an increase in activity in the 

female control group and decrease in activity in female mid exposure group only during week 

4.” Additional analysis of neurobehavioral endpoints was requested by EPA for 211(b) 

subchronic studies. That work was performed by ExxonMobil Biomedical Sciences (M. Nicolich 

and G. Bukhbinder) and used to write the draft report. The WG will instruct the study director to 

work with the contract statisticians to review and if appropriate clarify the text in the report.

Section 3.8 [p. 51] Clinical Pathology

Both reviewers requested more detailed discussions of statistical changes observed in 

hematology and clinical chemistry parameters.

Response: The biological significance of variable statistically significant changes in clinical 

pathology parameters in subchronic studies is difficult to determine in the absence of overt 
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physical effects or histologically observed tissue changes. The study duration is comparatively 

short and individual animal variability may produce wide standard deviations.

Section 3.8.1 [p. 51, Table 10] Hematology 

Dr. Goldsworthy:

Requested revision of reticulocyte count statements here and in the Summary (p. 7). He 

recommended that the designations „very slight” and “slight” be replaced with “statistical” 

decreases and that concluding statements (p. 7 and 55) should clarify that the decreases in 

reticulocyte counts occurred at all dose levels [in males] at week 4.

Response: The WG agrees that “slight” and “very slight” should be replaced with “statistical” 

unless there is an established quantified definition of these terms for “% change”. Although 

decreases in reticulocyte counts were observed in all male dose groups at week 4 as reported 

in the text (p. 51-52), only the 20000mg/m3 males showed consistent non-reversible 

occurrences at termination and after the recovery period warranting inclusion as a significant 

finding in the conclusions (p. 55) and Summary (p.7). No revisions are needed. 

Noted that the text did not address statistically significant (p<0.01) decrease in absolute 

monocyte counts in 20000mg/m3females at week 4 and at recovery.

Response: Although the statistically significant decrease in absolute monocyte counts in high 

dose females at week 4 and recovery (values at termination were lower than controls but were 

not statistically significant) are not likely to be biologically significant, the WG will instruct the 

study director to include this finding in the text with appropriate comment.

Section 3.8.2 [p. 52] Coagulation time

Dr. Schlesinger suggested that the increase in mean coagulation values in all animals exposed to 

BG+DIPE VC should be addressed.

Response: The cited increase in coagulation values occurred only in male rats at week 4. 

Increased prothrombin time and activated partial prothrombin time (APPT) were elevated 

above controls at approximately the same value for all doses at a significance level of 0.05 or 

0.01 respectively (PT: 11.9 sec. control vs 12.5, 12.6, 12.5 seconds for low, mid and high 

doses and APPT: 17.7 sec.control vs 20.4, 20.4, and 21.1 seconds for low, mid and high 

doses, respectively). At terminal sampling, values were similar to week 4 but were not 

statistically significant. The study director will add a description of this effect to the text but the 

change is likely a statistical anomaly rather than biologically significant in the absence of doserelated increases and no statistically significant value at the terminal evaluation.

Section 3.8.3 [p. 52] Clinical Chemistry

Both reviewers commented that this section does not adequately address the data and cite CK 

and SDH changes in males and SDH changes in females. Discussion appeared to focus on 

males only. Although Dr. Goldworthy agreed that “it is appropriate to not consider these clinical 

chemistry effects adverse”, he maintained the effects should be included in the results and the 

Summary.

Response: The WG agrees that additional information should be supplied in the Clinical 

Chemistry section. Expanded entries will be included on statistically significant changes in 

lactate dehydrogenase and sorbitol dehydrogenase levels in males and female animals, and 

increased levels of Na+ and K+ in female rats at all sampling intervals. The Summary section 

(p. 7) will be revised to include female data entries. However, the WG agrees with the study 

director that these changes are not consistent enough to be considered biologically significant 

effects of BG+DIPE VC exposure.
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Dr. Schlesinger was concerned that changes at week 4 that did not also occur at termination 

were not regarded as significant.

Response: Statistically significant changes that were observed only at week 4 in this study, 

included decreased glucose and creatinine kinase levels in males at all dose levels and bile 

acids (all groups), glucose (high dose) and alt (high dose) in females. In the absence of a 

persistent pattern of effect at these dose levels at termination and/or in high dose recovery 

animals, these early changes are considered transient adjustments to exposure that have no 

biologically significant effects.

Section 3.9 [p. 53] Organ Weights

Dr. Schlesinger pointed out a decrease in lung weight in males exposed at 20000mg/m3that 

should be included in the text.

Response: The text indicated that a few statistically significant differences in organ weights were 

observed but no details were given. The WG will advise that study director to include an entry 

that “a statistically significant decrease in absolute lung weight compared to controls was 

observed in 20000mg/m3exposed male rats but no statistical significance was found for lung 

weight relative to body weight or to brain weight, and no abnormal histopathology was 

observed.”

GFAP Assay [Dr. J. O’Callaghan]

Both reviewers agreed that the study was properly conducted and agreed with the conclusion 

that BG+DIPE VC did not cause gliosis in the brain regions examined. 

Dr. Schlesinger considered this a well written report and requested only that the name of the 

post-hoc test used be provided in the section on Statistics (p.17)

Response: The study director will request Dr. O‟Callaghan to supply this information.

Dr. Goldsworthy referred to earlier comments on the absence of study specifics and setting of 

criteria prior to study performance.

Response: The WG agrees with these comments. Dr. O‟Callaghan has been requested to 

expand on specific criteria to identify positive or suspect materials. However, widespread use 

of this assay is relatively recent and firm parameters to identify responses are still being 

established. It is also worth noting that Dr. O'Callaghan is internationally recognized for his 

work on the GFAP assay, and the WG has confidence in the integrity of the study methodology 

(including statistics) and its conclusions. 

Further, the WG has discussd with Dr. O‟Callaghan, deletion of sparse pituitary data from 

Tables 5 and 6, but retention of individual values in Table 7, primarily because this series of 

studies is one of the first in which GFAP levels in pituitary have been measured. This study 

had usable data only for 2 males and 1 female in the control group and three males in the 

20,000mg/m3 dose group. To include this limited data in summary tables (5&6) gives an 

erroneous impression that sufficient animals/group responded. Keeping individual entries 

(Table 7) provides an idea of the level of GFAP likely to be present in this small organ, 

providing a starting point for improvement of assay methods. 

Immunotoxicology Assay [Immunotox, Inc.]

Dr. Schlesinger inquired about the statement in the text that the “effect of DIPE on spleen 

weights and spleen cell numbers cannot be appropriately evaluated since the animals in this 

study were sensitized with sheep erythrocytes”. He wondered why these data are included if it 

is not possible to provide any conclusion.

Response: The statement in this report refers to comparison of these data with the spleen data 

in the 13-week study in which these animals were co-exposed but where no effects on spleen 

weight were reported. The overall increased relative spleen weights from animals induced with 
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S-RBC is not indicative of the effect of BG+DIPE VC on rats in general but is believed to be a 

useful measurement for immunologically related changes in this assay by the study director 

(Dr. White). The WG will not ask for changes in the report. 

Dr. Goldsworthy:

Requested that some historical control data be supplied to determine the significance of the 

decrease in spleen IgM response at the 10000mg/m3level compared to “normal vehicle 

controls”. The text [p. 16] had stated that vehicle control animal values were „somewhat lower 

than had been observed in the past.”

Response: Dr. White will be requested to supply historical vehicle control data for this 

comparison.

Suggested that the report conclusion on p. 9 and p. 17 provide a NOAEL = 10000mg/m3.

Response: Dr. White will be requested to consider adding a NOAEL to the report.

Micronucleus Assay [Huntingdon, UK]

Both reviewers agreed that the study was properly conducted and agreed with the conclusion 

that BG+DIPE VC did not cause an increased frequency of micronucleated immature 

erythrocytes in bone marrow of treated rats. No other comments were made.

Sister Chromatid Exchange Assay [BioReliance, Inc.]

Both reviewers agreed that the study was properly conducted and agreed with the conclusion 

that BG+DIPE VC did not induce sister chromatid exchanges (SCE) in rat peripheral blood. 

This study employed the statistics recommended by EPA.

Dr. Goldsworthy suggested that it be reported in the Summary that the positive control fulfilled 

the requirements for a valid test and that the study title should include Gasoline-DIPE Vapor 

Condensate.

Response: The WG will request the study director to include the statement about the positive 

control in the Summary. It does not seem necessary to change the title of the report since 

“Gasoline-DIPE Vapor Condensate” appears in the next line under Test Substance.

Although the results of this study are clearly negative, Dr. Schlesinger was concerned in general 

about some criteria used to evaluate SCE data. He did not agree that “a statistically significant 

increase in the high exposure level without statistically significant exposure level responses at 

lower doses should be considered suspect”. He suggested that this event should be 

considered a full positive response indicating that “some exposure threshold for response had 

been reached”.

Response: The SCE assay was developed as an indicator of DNA perturbation with possible 

application for suggesting a potential for chromosome or other genetic damage. Criteria for 

positive responses developed by many investigators evaluating numerous assays were 

designed to be sufficiently rigorous to minimize false positive and negative results in relation to 

other in vivo genetic assays and to strengthen predictability and reliability. The designation of 

“suspect” when statistical significance was observed only at the highest dose, and “equivocal” 

when positive results are observed at two doses in the absence of an exposure response trend 

contributed to improving this reliability by ranking the relevance of responses. The scientific 

expertise of the investigator is a significant factor in the accurate assessment of SCE data.